BRPI0717098B1 - ANALOGUE OF HUMAN INSULIN RESISTANT TO PROTEASE, PHARMACEUTICAL COMPOSITION AND PROCESS FOR PREPARATION - Google Patents

Description

(54) Title: PROTEASE RESISTANT HUMAN INSULIN ANALOGS, PHARMACEUTICAL COMPOSITION AND PROCESS FOR ITS PREPARATION (51) Int.CI .: C07K 14/62; A61K 38/28; C12N 15/17 (30) Unionist Priority: 22/09/2006 EP 06 121113.2 (73) Holder (s): NOVO NORDISK A / S (72) Inventor (s): PETER KRESTEN NIELSEN; FRANTISEK HUBALEK; INGER LAUTRUP-LARSEN; SVEND LUDVIGSEN; ULLA RIBEL-MADSEN; PER BALSCHMIDT; PER N0RGAARD; SVEND HAVELUND (85) National Phase Start Date: 23/03/2009

Invention Patent Descriptive Report for PROTEASE-RESISTANT HUMAN INSULIN ANALOGS, PHARMACEUTICAL COMPOSITION AND PROCESS FOR ITS PREPARATION.

Field of the Invention

The present invention relates to new insulin analogs that exhibit resistance to protease, to a method for the preparation of such insulin analogs, to insulin preparations containing the insulin analogs of the invention and to a method of treating diabetes mellitus using these insulin analogs.

Background of the Invention

The oral route is by far the most widely used route for drug administration and is in general very well tolerated by patients, especially for chronic therapies. The administration of therapeutic peptides or proteins is, however, often limited to parenteral routes rather than the preferred oral administration due to various barriers, such as enzymatic degradation in the gastrointestinal (GI) tract, drug efflux pumps, insufficient absorption and intestinal mucosa, as well as first-pass metabolism in the liver. Human insulin is degraded by various digestive enzymes found in the stomach (pepsi20 na), in the intestinal lumen (chymotrypsin, trypsin, elastase, carboxypeptidases, etc.) and on mucous surfaces of the GI tract (aminopeptidases, carboxypeptidases, enteropeptidases, dipeptidil peptidases, endopeptidases , etc.).

This is unfortunate because many peptides and proteins have proven to be clinically effective and could be more widely used if easy to administer and acceptable to recipients.

Diabetes mellitus is a metabolic disorder in which the ability to use glucose is partly or completely lost. Approximately 5% of all people suffer from diabetes and the disorder is approaching epidemic proportions. Since the introduction of insulin in the

1920, continuous efforts were made to improve the treatment of diabetes mellitus. As people suffering from diabetes have been subject to chronic treatment for several decades, there is a great need

Petition 870170075355, of 10/05/2017, p. 9/27 of safe, adequate insulin formulations that improve quality of life.

In the treatment of diabetes mellitus, many varieties of insulin formulations have been suggested and used, such as regular insulin, isophane insulin (called NPH), zinc insulin suspension (such as Semilente®, Lente® and Ultralente®), and isophane insulin biphasic. Some of the commercially available insulin formulations are characterized by a rapid onset of action and other formulations have a relatively slow onset, but show a more or less prolonged action. Fast-acting insulin formulations are usually insulin solutions, whereas delayed-acting insulin formulations can be suspensions containing crystalline and / or amorphous insulin precipitated only by the addition of zinc salts or the addition of protamine or a combination both.

Human insulin is composed of two polypeptide chains, chains A and B that contain 21 and 30 amino acid residues, respectively. Chains A and B are linked by two disulfide bridges. Insulin in most other species is similar, but may contain amino acid substitutions in some positions. In the past decade, several human insulin analogs have been developed. They are designed for certain action profiles, that is, fast acting or long acting. Commercially available products comprising such insulin analogs include Levemir®, NovoRapid®, Humalog®, Apidra® and Lantus®.

Usually, insulin formulations are administered via subcutaneous injection.

However, oral administration would be advantageous due to adaptation, safety and convenience for the patient.

Oral administration of protein drugs, such as insulin, often results in very low bioavailability due to enzyme and absorption barriers. The general approach to the release of the peptide and protein is parenteral administration which is invasive and inconvenient. Therefore, non-invasive pathways such as pharmaceutical products based on the oral release of the protein are increasingly investigated. Recent formulation designs for oral protein / peptide release include co-formulations with protease inhibitors, permeation enhancers, polymer-based delivery systems and insulin conjugates. The latter includes hexyl-insulin-monoconjugate-2 (HIM2) (Nobex Cooperation and GSK), a human insulin analog with a 7-hexyl PEG group attached to B29. For example, US 7,030,082, US 6,867,183 and US 6,770,625, oral HIM2 has been reported to have proteolytic stability and increased bioavailability compared to insulin.

Summary of the Invention

In an embodiment of the invention an insulin analogue with enhanced proteolytic stability and preserved insulin biological activity is provided.

In a further embodiment of the invention, an insulin analog is provided in which at least two amino acids are substituted and / or deleted with respect to the precursor insulin molecule.

In a further embodiment of the invention, an insulin analogue is provided in which at least two hydrophobic amino acids have been replaced with hydrophilic amino acids in relation to the precursor insulin, where the substitutions are within or in the vicinity of two or more protease dividing sites. of the precursor insulin and wherein such an insulin analogue optionally still comprises one or more additional mutations.

It is also an object of the invention to provide insulin analogs according to the invention in which the A chain of the insulin analog comprises at least one mutation and the B chain of the insulin analog comprises at least one mutation and, in relation to the precursor insulin.

The present invention also relates to nucleic acid sequences that code for the pre-propeptides of the claimed insulin analogs. In a further embodiment, the present invention relates to vectors that contain such nucleic acid sequences and host cells that contain such nucleic acid sequences or vectors.

A process for obtaining an insulin analog, according to the invention, and the use thereof for use as a pharmaceutical product is also provided.

Description of the Figures

Figurei: proteolytic stability of human insulin (IH) and insulin analogues in relation to chymotrypsin, measured as percentage of intact (analogous) insulin at 37 ° C.

Figure 2: Proteolytic stability of human insulin (IH) and insulin analogues in relation to pepsin, measured as percentage of intact (analogous) insulin at 25 ° C.

Description of the Invention

An insulin analogue according to the invention is an insulin molecule that has two or more mutations of the amino acid chains A and / or B with respect to the precursor insulin molecule.

It has been found that by replacing two or more hydrophobic amino acids within or in the vicinity of two or more protease sites on an insulin with hydrophilic amino acids, an insulin analogue is obtained which is the proteolytically stable compared to the precursor insulin.

The following is a non-restrictive list of modalities, which is further described elsewhere in this application.

Mode 1. An insulin analogue in which at least two hydrophobic amino acids have been replaced with hydrophilic amino acids in relation to the precursor insulin, where the substitutions are within or in the vicinity of two or more protease dividing sites of the precursor insulin and where such insulin analog optionally still comprises one or more additional mutations.

Mode 2. An insulin analogue according to mode 1 in which increased solubility in relation to the precursor insulin is obtained.

Mode 3. An insulin analogue according to any one of modalities 1 to 2 wherein the insulin analog A chain comprises at least one mutation and the insulin analog B chain comprises at least one mutation with respect to the precursor insulin.

Mode 4. An insulin analogue according to any one of modalities 1 to 3 wherein the insulin analog further comprises at least one amino acid substitution at a protease site for a first modified insulin analogue, where at least one said amino acid substitution is such that at least one hydrophobic amino acid has been replaced with at least one hydrophilic amino acid.

Modality 5. An insulin analog according to any of modalities 1 to 4 where · Ο amino acid at position A12 is Glu or Asp and / or the amino acid at position A13 is His, Asn, Glu or Asp and / or the amino acid in position A14 is Asn, Gin, Glu, Arg, Asp, Gly or His and / or the amino acid at position A15 is Glu or Asp; and • The amino acid at position B24 is His and or the amino acid at position B25 is His and / or the amino acid at position B26 is His, Gly, Asp or Thr and / or the amino acid at position B27 is His, Glu, Lys, Gly or Arg and / or the amino acid at position B28 is His, Gly or Asp; and that optionally still comprises one or more additional mutations.

Mode 6. An insulin analogue according to any one of modalities 1 to 5 in which the amino acid at position A14 is Glu, Asp or His, the amino acid at position B25 is His and which optionally still comprises one or more additional mutations.

Mode 7. An insulin analogue according to any of modalities 1 to 6 in which the amino acid at position A14 is Glu, Asp or His, the amino acid at position B25 is His and the amino acid at position B30 is deleted.

Mode 8. An insulin analogue according to any of modalities 1 to 6 in which the amino acid at position A14 is Glu, Asp or His and the amino acid at position B25 is His.

Mode 9. An insulin analogue according to any one of modalities 1 to 6 in which one or more additional mutations are selected from a group consisting of: A (-3) Gly, A (-2) Gly, A (-1 ) Pro, A (0) Pro, A8His, A18Gln, A18Gln, A21Gln, A21Gly, B (-3) Gly, B (-2) Gly, B (1) Pro, B (0) Pro, B1Glu, B1Gln, ro , B1Glu, B1Gln, B3Gln, B10Pro, B14Thr, B16Glu, B17Ser, B26Asp, DesB26, DesB27, B27Glu, B27Glu, B28Asp, desB28, desB29, desB30, B31Leu, B32Glu.

Mode 10. An insulin analogue according to any one of modalities 1 to 6 or 9 where the additional mutation is desB30.

Mode 11. An insulin analogue according to any of modes 1 to 10 where A14 is Glu.

Mode 12. An insulin analogue according to any of modalities 1 to 11 that shows increased stability in relation to one or more protease enzymes in relation to the precursor protein.

Mode 13. An insulin analogue according to any one of modalities 1 to 12 that shows increased stability in relation to two or more protease enzymes, in relation to the precursor protein.

Mode 14. An insulin analogue according to any of modes 1 to 13 in which the precursor insulin is selected from a group consisting of:

• human insulin;

· An insulin analogue of human insulin in which the amino acid residue at position B28 is Pro, Asp, Lys, Leu, Vai or Ala and the amino acid residue at position B29 is Lys or Pro and optionally the amino acid residue at position B30 is deleted;

• human insulin (B26-B30), human insulin (B2725 B30), human insulin (B28-B30), human insulin (B29-B30), human insulin (B27) or human insulin (B30);

• an insulin analog of human insulin in which the amino acid residue at position B3 is Lys and the amino acid residue at position B29 is Glu or Asp;

· An insulin analog of human insulin in which the amino acid residue at position A21 is Gly and in which the insulin analog is further extended at Terminal C with two Arg residues;

• an insulin derivative in which the amino acid residue at position B30 is replaced with a threonine methyl ester; and • an insulin derivative in which the 29ε position of lysine at position B29 of the (B30) human insulin is attached to a tetradecanoyl chain.

Mode 15. An insulin analogue according to any of the modalities 1 to 6, 9 or 11 to 14 in which one or more additional mutations are selected to enhance the chemical stability of insulin.

Mode 16. An insulin analogue according to mode 15 in which one or more additional mutations are selected from a group consisting of: A18Gln, A21Gln, A21GLy and B3Gln.

Mode 17. An insulin analogue according to any one of modalities 1 to 4 or 12 to 14 comprising an A chain amino acid sequence of formula 1:

XaaA (-2) -Xaa _{A (} .-I) -XaaAo-Gly-He-Val-Glu-Gln-Cys-Cys-XaaA8-SerHe-Cys-Xaa _A i2-XaaAi3-XaaAi4-XaaAi5-Leu-Glu- XaaAi8-Tyr-Cys-XaaA2i-XaaA22

Formula (1) (SEQ ID NO: 1) and a sequence of B chain amino acids of formula 2:

Xaa _B (-2) -XaaB (-i) -XaaBo-Xaa _B1 -XaaB2-XaaB3-XaaB4-His-Leu-CysGly-Ser-Xaa _B1 o-Leu-Val-Glu-Ala-Leu-XaaBi6-Leu- Val-Cys-Gly-Glu-Arg-GlyXaa _B 24-XaaB25-XaaB26-XaaB27-XaaB28-XaaB29-Xaa _B 30-XaaB3i-XaaB32

Formula (2) (SEQ ID NO: 2) where

Xaa _A (-2) θ absent or Gly;

Xaa _A (-i) is absent or Pro;

Xaa _A o θ absent or Pro;

Xaa _A 8 is independently selected from Thr and His;

Xaa _A i2 θ independently selected from Ser, Asp and

Glu;

Xaa _A i3 is independently selected from Leu, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

Xaa _A i4 is independently selected from Tyr, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

Xaa _A i5 is independently selected from Gin, Asp and

Glu;

Xaa _A is is independently selected from Asn, Lys and

Gin;

Xaa _A2 i is independently selected from Asn and Gin; Xaa _A2 2 θ absent or Lys;

Xaa _B (-2) is absent or Gly;

Xaa _B (-η is absent or Pro;

Xaa _B0 is absent or Pro;

Xaa _B i is absent or is independently selected from Phe and Glu;

Xaa _B2 is absent or Vai;

Xaa _B 3 is absent or is independently selected from Asn and Gin;

Xaa _B4 is independently selected from Gin and Glu; Xaa _B w is independently selected from His, Asp,

Pro and Glu;

Xaa _B16 is independently selected from Tyr, Asp, Gin, His, Arg, and Glu;

Xaa _B2 4 is independently selected from Phe and His; Xaa _B2 5 θ independently selected from Phe and His; Xaa _B 26 is absent or is independently selected from Tyr, His, Thr, Gly and Asp;

Xaa _B2 7 is absent or is independently selected from Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

Xaa _B2 8 is absent or is independently selected from Pro, His, Gly and Asp;

Xaa _B2 9 is absent or is independently selected from Lys and Gin;

Xaa _B30 is absent or Thr;

Xaa _B3 i is absent or Leu;

Xaa _B32 is absent or Glu;

Terminal C can optionally be derivatized as an amide;

wherein the A chain amino acid sequence and the B chain amino acid sequence are joined by disulfide bridges between the cysteines at position 7 of the A chain and the cysteine at position 7 of the B chain, and between the cysteine at position 20 of the chain A and cysteine at position 19 of chain B and where cysteines at position 6 and 11 of chain A are joined by a disulfide bridge;

wherein the N-terminal A chain amino acid sequence is optionally joined to the C-terminal B chain amino acid sequence by an amino acid sequence comprising 3 to 7 amino acids for forming a single-chain insulin molecule, where the Terminal optionally B chain N is extended with 1 to 10 amino acids;

where if XaaAe θ Thr and XaaAi2 θ Ser and Xaa _A i3 is Leu and Xaa _A i4 θ Tyr, then Xaa _A i5 is Glu or Asp; and where if Xaa _B24 is Phe and Xaa _B25 is Phe and Xaa _B2 and is Tyr and Xaa _B27 is Thr and Xaa _B2 e is Pro, then Xaa _B29 Gin.

Method 18. An insulin analogue according to any of modalities 1a4 or 12a14 comprising an A chain amino acid sequence of formula 3:

Gly-Ile-Val-Glu-Gln-Cys-Cys-XaaA8-Ser-Ile-Cys-Xaa-XaaAi2 _The i3Xaa _The i4-XaaAi5-Leu-Glu-Cys-Tyr-XaaAi8-Xaa _The 2i

Formula (3) (SEQ ID NO: 3) and a B chain amino acid sequence of formula 4:

Xaa _B i-Val-XaaB ₃ -Xaa _B4 -His-Leu-Cys-Gly-Ser-Xaa _B io-Leu-ValGlu-Ala-Leu-Xaa _B i ₆ -Leu-Val-Cys-Gly-Glu-Arg -Gly-Xaa _B2 4-His-Xaa _B26 -Xaa _B27 Xaa _{B2 8} -Xaa _B2 g-Xaa _B 3

Formula (4) (SEQ ID NO: 4) where

Xaa _A and θ independently selected from Thr and His;

XaaAi2 θ independently selected from Ser, Asp and

Glu;

XaaAn θ independently selected from Leu, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

Xaa _A i4 is independently selected from Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

XaaAis θ independently selected from Gin, Asp and

Glu;

XaaAie θ independently selected from Asn, Lys and

Gin;

Xaa _A 2i θ independently selected from Asn, and Gin; Xaaei is independently selected from Phe and Glu; Xaa _B3 is independently selected from Asn and Gin; Xaa _B4 θ independently selected from Gin and Glu; Xaa _B10 is independently selected from His, Asp,

Pro and Glu;

Xaa _B i6 θ independently selected from Tyr, Asp, Gin, His, Arg, and Glu;

Xaa _B2 4 θ independently selected from Phe and His; Xaa _B 26 is absent or independently selected from

Tyr, His, Thr, Gly and Asp;

Xaa _B 27 is absent or independently selected from Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

Xaa _B 28 is absent or is independently selected from Pro, His, Gly and Asp;

Xaa _B 29 θ absent or independently selected from

Lys and Gin;

Xaa _B 3 is absent or Thr;

Terminal C can optionally be derivatized as an amide;

where the amino acid sequence of chain A and the amino acid sequence of chain B are disulfide bonded between the cysteines at position 7 of chain A and the cysteine at position 7 of chain B, and between cysteine at position 20 of chain A and cysteine at position 19 of chain B and where the cysteines at position 6 and 11 of chain A are joined by a disulfide bridge.

Mode 19. An insulin analogue according to mode 18, where

Xaa _A 8 θ independently selected from Thr and His; Xaa _A i2 is independently selected from Ser and Glu; Xaa _A13 is independently selected from Leu, Thr,

Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser and Glu;

Xaa _A14 is independently selected from Asp, His, and

Glu;

Xaa _A i5 is independently selected from Gin and Glu; Xaa _A18 is independently selected from Asn, Lys and

Gin;

Xaa _A 2i is independently selected from Asn, and Gin; Xaa _B i is independently selected from Phe and Glu; Xaa _B3 is independently selected from Asn and Gin; Xaa _B4 is independently selected from Gin and Glu; Xaa _B w is independently selected from His, Asp,

Pro and Glu;

Xaa _B i6 is independently selected from Tyr, Asp, Gin, His, Arg, and Glu;

Xaa _B 24 is independently selected from Phe and His; Xaa _B 26 is independently selected from Tyr, Thr,

Gly and Asp;

Xaa _B 27 is independently selected from Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, and Glu;

Xaa _B28 is independently selected from Pro, Gly and

Asp;

Xaa _B2 9 is independently selected from Lys and Gin;

Xaa _B 3 θ absent or Thr;

Terminal C can optionally be derivatized as an amide;

wherein the chain A amino acid sequence and chain B amino acid sequence are linked by disulfide bridges between the cysteines at position 7 of chain A and the cysteine at position 7 of chain B, and between cysteine at position 20 of chain A and cysteine at position 19 of chain B and where cysteines at position 6 and 11 of chain A are joined by a disulfide bridge.

Mode 20. An insulin analogue according to any one of modalities 1 to 16 in which the B-chain Terminal C is joined to the A-terminal chain N with 3 to 15 amino acids or 3 to 7 amino acids, to form a single-chain insulin molecule , where optionally the N-terminal of the B chain is extended with 1 to 10 amino acids.

Mode 21. A pharmaceutical composition comprising a biologically active amount of the insulin analogue according to any one of modalities 1 to 20 and a pharmaceutically acceptable carrier.

Mode 22. A pharmaceutical composition comprising two or more insulin analogs, according to any one of modalities 1 to 20 in which each analogue is defined to have at least one mutation, which is absent in at least one of the other variants.

Mode 23. A pharmaceutical composition according to any one of modalities 11 to 20 which further comprises an acceptable pharmaceutical excipient and / or vehicle, and optionally an adjuvant.

Mode 24. A method for the treatment of diabetes mellitus in an individual comprising administering to an individual an insulin analogue according to any one of modalities 1 to 20 or a pharmaceutical composition according to any one of modalities 21 to 23 .

Mode 25. A method for reducing the blood glucose level in mammals by administering to a patient in need of such treatment a therapeutically active dose of an insulin analogue according to any one of modalities 1 to 20 or a pharmaceutical composition according to with any of the modalities 21 to

23.

Modality 26. Method according to modality 24 or 25 being an oral administration.

Modality 27. Method according to modality 24 or 25 being parenteral administration.

Modality 28. Method according to modality 24 or 25 being intratracheal administration.

Mode 29. An insulin analogue according to any of modalities 1 to 20 for use as a pharmaceutical product in the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, syndrome X and dyslipidemia.

Mode 30. An insulin analogue according to any one of modalities 1 to 20 for use as a pharmaceutical product slowing or preventing disease progression in type 2 diabetes.

Mode 31. An insulin analogue according to any of modalities 1 to 20 for use as a pharmaceutical product in reducing food intake, reducing β cell apoptosis, increasing β cell function and increasing number of β cells and / or to restore the sensitivity of β cells to glucose.

Mode 32. A nucleic acid sequence encoding an insulin analogue according to any one of modalities 1 to 20, a derivative thereof, a partial sequence thereof, a degenerate sequence thereof or a sequence that hybridizes even under conditions stringent.

Mode 33. A nucleic acid sequence that encodes a precursor to an insulin analogue according to any one of modalities 1 to 20, a derivative thereof, a partial sequence thereof, a degenerate sequence thereof or a sequence that hybridizes even under strict conditions.

Mode 34. An expression vector comprising a nucleic acid sequence according to mode 32 or 33.

Modality 35. A host cell comprising an expression vector according to modality 34.

Mode 36. A method for producing an insulin analog comprising the stage of culturing the host cell of mode 35.

Mode 37. A method for preparing an insulin analogue according to any one of modalities 1 to 20 in which the amino acid substitution is carried out by site-directed mutagenesis.

Mode 38. A process for preparing a pharmaceutical composition according to any of modalities 21 to 23 comprising mixing an insulin analogue according to any of modalities 1 to 20 with pharmaceutically acceptable substances and / or excipients.

Mode 39. A pharmaceutical composition obtainable through the process according to mode 38.

Insulin is a polypeptide hormone secreted by β cells in the pancreas. Insulin is composed of two polypeptide chains, A and B, which are linked by two interchain disulfide bridges. In addition, chain A has an intra-chain disulfide bridge.

The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) composed of a 24-amino acid pre-peptide followed by a pro-insulin containing 86 amino acids in the configuration: pre-peptide-B-Arg Arg-C-Lys Arg -A, in which C is a 31 amino acid binding peptide. Arg-Arg and Lys-Arg are dividing sites for dividing the A and B chain binding peptide.

The insulin analogs according to the invention can comprise additional mutations. A mutation in an insulin molecule can be in the form of a substitution, a deletion or an addition of an amino acid residue in the A and / or B chain of the naturally occurring insulin molecule.

With desB30 or “B (1-29)” it is understood that the B chain of natural insulin loses the amino acid residue B30 and “A (1-21)” means the A chain of natural insulin. The mini peptide C and its amino acid sequence are indicated in the three letter code of amino acids.

In this order terms such as A1, A2, A3 etc. indicate position 1, 2 and 3, respectively, in the A insulin chain (counted from the final N-Terminal). Similarly, terms like B1, B2, B3 etc. indicate position 1, 2 and 3, respectively, in the insulin B chain (counted from the final N-Terminal). Using the one letter code for amino acids, terms like A21A, A21G and A21Q indicate that the amino acid at position A21 is A, G and Q, respectively. Using the three letter code for amino acids, the corresponding expressions are A21Ala, A21Gly and A21Gln, respectively.

In this order, the terms A (0) or B (0) indicate the Terminal positions Nearly A1 or B1, respectively. The terms A (-1) or B (1) indicate the positions of the first Terminal amino acids to A (0) or B (0), respectively. Thus A (-2) and B (-2) indicate positions N-terminals A (-1) and B (-1), respectively, A (-3) and B (-3) indicate positions Terminal Nmente to A (- 2) and B (-2), respectively, and so on.

The term binding peptide encompasses a peptide chain that can join the B-terminal C amino acid residue with the A-chain N terminal amino acid residue on insulin.

The term pro-peptide means a polypeptide sequence whose function must allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and to a secretory vesicle for secretion in the culture medium (that is, the export of the polypeptide through the cell wall or at least by the cell membrane in the periplasmic space of the yeast cell). The pro-peptide may be the yeast α-factor pro-peptide see US 4,546,082 and 4,870,008. Alternatively, the pro-peptide can be a synthetic pro-peptide, that is, a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in US 5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The propeptide will preferably contain an endopeptidase processing site at the end of Terminal C, such as a Lys-Arg sequence or any functional analog thereof.

The term diabetes includes type 1 diabetes, type 2 diabetes and other conditions that cause hyperglycemia.

The term "treatment" of a disease includes the treatment, prevention or alleviation of the disease.

In an embodiment of the invention, the insulin analog is especially suitable for oral administration.

Hydrophobic amino acids are to be understood in this application as naturally occurring amino acids tryptophan (Trp, W), phenylalanine (Phe, F), valine (Vai, V), isoleucine (lie, I), leucine (Leu, L) and tyrosine (Tyr , Y) (with the abbreviation of three letters and one letter in parentheses).

Hydrophilic amino acids are to be understood in this application as natural amino acids that are not hydrophobic amino acids according to the definition above. In one embodiment, hydrophilic acids according to the invention are selected from the group consisting of; glutamic acid (Glu, E), aspartic acid (Asp, D), histidine (His, H), glutamine (Gin, Q), asparagine (Asn, N), serine (Ser, S), threonine (Thr, T) , proline (Pro, P), glycine (Gly, G), lysine (Lys, K) and arginine (Arg, R). In an additional embodiment, hydrophilic amino acids according to the invention are selected from the group consisting of: glutamic acid (Glu, E), aspartic acid (Asp, D), histidine (His, H), glutamine (Gin, Q) , asparagine (Asn, N), lysine (Lys, K) and arginine (Arg, R).

"An insulin" according to the invention is to be understood in this application as human insulin, an insulin analogue or an insulin derivative.

The term precursor insulin, as used in this application, is intended to mean an insulin before any mutations according to the invention have been applied to it. Unlimited examples of precursor insulin are, for example, a wild-type insulin, such as human insulin or porcine insulin, a human insulin analogue or a derivative of human insulin, or an insulin analogue, such as human insulin or an insulin analogue. insulin that has been PEGylated or acylated.

In one embodiment, a precursor insulin according to the invention is selected from the group consisting of:

human insulin;

an insulin analog of human insulin in which the amino acid residue at position B28 is Pro, Asp, Lys, Leu, Vai, or Ala and the amino acid residue at position B29 is Lys or Pro and optionally the amino acid residue at position B30 is deleted;

an insulin analog which is des human insulin (B28-B30), des human insulin (B27) or des human insulin (B30);

an insulin analog of human insulin in which the amino acid residue at position B3 is Lys and the amino acid residue at position B29 is Glu or Asp;

an insulin analog of human insulin in which the amino acid residue at position A21 is Gly and in which the insulin analog is further extended at Terminal C with two arginine residues;

an insulin derivative in which the amino acid residue at position B30 is replaced with a threonine methyl ester; and an insulin derivative in which the inaε position of lysine is attached to the B29 position of human des (B30) insulin a tetradecanoyl chain.

In one embodiment, a precursor insulin according to the invention is selected from the group consisting of:

human insulin; desB30 human insulin; human insulin AspB28; human insulin AspB28, desB30; human insulin LysB3, GluB29; human insulin LysB28, ProB29; human insulin GlyA21, ArgB31, ArgB32; and desB30, ArgB31, ArgB32 human insulin.

The term insulin analogue as used in this application means a modified insulin in which one or more amino acid residues of the insulin have been replaced by others of amino acid residues and / or in which one or more amino acid residues have been deleted from the insulin and / or in that one or more amino acid residues have been added to insulin.

In one embodiment, an insulin analog comprises less than 8 changes (substitutions, deletions, additions) in relation to the precursor insulin. In one embodiment, an insulin analog comprises less than 7 modifications (substitutions, deletions, additions) in relation to the precursor insulin. In one embodiment, an insulin analog comprises less than 6 modifications (substitutions, deletions, additions) in relation to the precursor insulin. In another embodiment, an insulin analog comprises less than 5 changes (substitutions, deletions, additions) in relation to the precursor insulin. In another embodiment, an insulin analog comprises less than 4 changes (substitutions, deletions, additions) in relation to the precursor insulin. In another embodiment, an insulin analogue comprises less than 3 changes (substitutions, deletions, additions) in relation to the precursor insulin. In another embodiment, an insulin analogue comprises less than 2 changes (substitutions, deletions, additions) in relation to the precursor insulin.

The term insulin derivative as used in this application means a chemically modified precursor insulin or even analogues thereof, wherein at least one substituent is not present in the precursor protein or analog thereof, that is, a precursor protein that has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, PEGylations and the like. Examples of human insulin derivatives according to the invention are human insulin B30 threonine methyl ester, human insulin des (B30) Ne-B29-tetradecanoyl, human insulin GlnB3 des (B30) Ne-B29-tetradecanoyl, human insulin N £ - B29-tridecanoyl, human insulin Ne-B29-tetradecanoyl, human insulin Ne-B29-decanoyl and human insulin Ne-B29-dodecanoyl

The term human insulin as used in this application means the human hormone whose structure and properties are well known. Human insulin has two polypeptide chains that are linked by disulfide bridges between cysteine residues, that is, chain A and chain B. Chain A is a 21-amino acid peptide and chain B is a 30-amino acid peptide, the two chains being joined by three disulfide bridges: one between cysteines at position 6 and 11 of chain A, the second between cysteine at position 7 of chain A and cysteine at position 7 of chain B, and the third between cysteine at position 20 of Chain A and cysteine at position 19 of Chain B.

Mutations in the insulin molecule are denoted by determining the chain (A or B), the position, and the three-letter amino acid code that replaces the native amino acid. DesB30 means a natural B-chain insulin or an analogue of the same that has lost the amino acid B30. Thus, human insulin A21Gly, B28Asp, desB30 is an analogue of human insulin where position 21 on a chain is mutated to glycine, position 28 on chain B is mutated to aspartic acid, and position 30 on chain B is deleted .

A protease or a “protease enzyme” is a digestive enzyme that degrades protein and peptides and is found in various tissues of the human body such as, for example, the stomach (pepsin), the intestinal lumen (chymotrypsin, trypsin, elastase, carboxypeptidases , etc.) or mucosal surfaces of the GI tract (aminopeptidases, carboxypeptidases, enteropeptidases, dipeptidyl peptidases, endopeptidases, etc.), the liver (insulin-degrading enzyme, cathepsin D etc.), and in other tissues.

A proteolytically stable insulin analog should be understood in this application as an insulin analog, which is subjected to the slowest degradation by one or more proteases in relation to human insulin. In one embodiment, a proteolytically stable insulin analogue according to the invention is subjected to the slowest degradation by one or more proteases in relation to the precursor insulin. In a further embodiment of the invention, an insulin analogue according to the invention is stabilized against degradation by one or more enzymes selected from the group consisting of: pepsin (such as, for example, the isoforms pepsin A, pepsin B, pepsin C and / or pepsin F), chymotrypsin (such as, for example, the chymotrypsin A, chymotrypsin B and / or chymotrypsin C isoforms), trypsin, insulin-degrading enzyme (IDE), elastase (such as, for example, pancreatic elastase isoforms I and / or II), carboxypeptidase (for example, carboxypeptidase A, carboxypeptidase A2 and / or carboxypeptidase B), aminopeptidase, cathepsin D and other enzymes present in intestinal extracts derived from rat, pig or human.

In one embodiment, an insulin analogue according to the invention is stabilized against degradation by one or more enzymes selected from the group consisting of: chymotrypsin, trypsin, insulin-degrading enzyme (IDE), elastase, carboxypeptidases, aminopeptidases and cathepsin D. In an additional embodiment, an insulin analogue according to the invention is stabilized against degradation by one or more enzymes selected from the group consisting of: chymotrypsin, carboxypeptidases and IDE. In an additional embodiment, an insulin analogue according to the invention is stabilized in relation to degradation by one or more enzymes selected from: chymotrypsin and carboxypeptidases.

T% can be determined as described in the Examples as a measure of the proteolytic stability of an insulin analog, according to the invention, in relation to protease enzymes, such as chymotrypsin, carboxypeptidase and / or pepsin A. In an embodiment of the invention T % is increased in relation to human insulin. In an additional modality, T% is increased in relation to the precursor insulin. Still in an additional modality, 1½ is increased at least 2 times compared to the precursor insulin. Still in an additional modality, T% is increased at least 3 times in relation to precursor insulin. Still in an additional modality, P / 2 is increased at least 4 times in relation to the precursor insulin. Still in an additional modality, P / 2 is increased at least 5 times in relation to the precursor insulin. Still in an additional modality, T / 2 is increased at least 10 times in relation to the precursor insulin.

Protease dividing sites (in this application also referred to as protease sites) are to be understood as amino acid residues that are recognized by amino acid residues and / or proteases whose peptide bond is cleaved by proteases. Protease divage sites can be defined by determining divage “hot spots” analyzed by HPLC, MS or LC-MS and / or by prediction based on the enzyme specificity of the protease enzyme for which the protease divage site should be determined. One skilled in the art will know how to determine protease dividing sites, for example, based on enzyme specificities as, for example, described in the Handbook of Proteolytical Enzymes, 2nd ed., Barrett, AJ, Rawlings, ND, Woesner, JF editors, Elsevier Academic Press 2004. For example, chymotrypsin is predicted to cleave Terminal C peptide bonds to aromatic residues (Trp, Tyr, Phe or Leu), which are not followed by Pro. Similarly, trypsin is predicted to cleave Terminal peptide bonds C to basic Lys or Arg residues, which are not followed by Pro, elastase is predicted to cleave Terminal C from Ala, Vai, Gly or Ser residues and carboxypeptidase A will remove any amino acid from Terminal C, but not Arg, Lys or Pro. The insulin-degrading enzyme (IDE) is predicted to cleave the following positions of human insulin B9-10, B10-11, B13-14, B14-15, B24-25, B25-26, A13-14 and A14-15 .

The term replacing (one) amino acid "within or near" a protease dividing site is used in this application to indicate the replacement of amino acids within or near a precursor insulin position that has been determined to be a dividing site protease. In one embodiment two or more hydrophobic amino acids within or in the vicinity of two or more protease sites in an insulin are replaced, in which said hydrophobic amino acids are replaced with hydrophilic amino acids. In an additional embodiment, two or more hydrophobic amino acids within two or more protease sites on an insulin are replaced with hydrophilic amino acids. Still in an additional embodiment, two or more hydrophobic amino acids located next to two or more protease sites on an insulin are replaced with hydrophilic amino acids. Still in an additional embodiment, two or more hydrophobic amino acids located two amino acids away from two or more protease sites in an insulin are replaced with hydrophilic amino acids. Still in an additional embodiment, two or more hydrophobic amino acids located three amino acids away from two or more protease sites in an insulin are replaced with hydrophilic amino acids. In an additional modality, two or more hydrophobic amino acids located up to four amino acids away from two or more protease sites in an insulin are replaced with hydrophilic amino acids. Still in an additional embodiment, two or more hydrophobic amino acids located one, two, or three amino acids apart or within two or more protease sites in an insulin are replaced with hydrophilic amino acids. In an additional embodiment, two or more hydrophobic amino acids located one or two amino acids apart or within two or more protease sites in an insulin are replaced with hydrophilic amino acids. In an additional modality, two or more hydrophobic amino acids located next to or within two or more protease sites in an insulin are replaced with hydrophilic amino acids.

An insulin analogue according to the invention can have a net charge that is different from the net charge of the precursor insulin. In one embodiment, the net charge of an insulin analog according to the invention is more positive than the net charge of the precursor insulin. In one embodiment, the net charge of an insulin analog according to the invention is more negative than the net charge of the precursor insulin. In one embodiment, the average positive net charge of an insulin analog, according to the invention, is between 0.5 and 5 as measured in an aqueous solution. In one embodiment, the average positive net charge of an insulin analog according to the invention is between 1 and 5. In one embodiment, the average positive net charge of an insulin analog according to the invention is between 1 and 4. In one embodiment, the average positive net charge of an insulin analogue according to the invention is between 1 and 3. In one embodiment, the average positive net charge of an insulin analogue according to the invention is between 2 and 3 In one embodiment, the average negative net charge of an insulin analog according to the invention is between -0.5 and -5 as measured in an aqueous solution. In one embodiment, the average negative net charge of an insulin analog according to the invention is between -1 and -5. In one embodiment, the average negative net charge of an insulin analog according to the invention is between -1 and -4. In one embodiment, the average negative net charge of an insulin analog according to the invention is between -1 and -3. In one embodiment, the average negative net charge of an insulin analog according to the invention is between -2 and -3.

In one embodiment, an insulin analogue according to the invention may have increased solubility over human insulin. In an additional embodiment, an insulin analog, according to the invention, had increased solubility in relation to human insulin at pH 3 to 9. Still in an additional embodiment, an insulin analog, according to the invention, had the increased solubility compared to human insulin at pH 4 to 8.5. Still in an additional embodiment, an insulin analogue, according to the invention, had the solubility increased in relation to human insulin at pH 4 to 8. Still in an additional embodiment, an insulin analogue according to the invention had the solubility increased in relation to human insulin at pH 4.5 to 8. In an additional embodiment, an insulin analog, according to the invention, had increased solubility in relation to human insulin at pH 5 to 8. Still in an additional embodiment , an insulin analog, according to the invention, had the solubility increased compared to human insulin at pH 5.5 to 8. In an additional embodiment, an insulin analog, according to the invention, had the solubility increased by relation to human insulin at pH 6 to 8.

In one embodiment, an insulin analog, according to the invention, had increased solubility compared to human insulin at pH 2 to 4.

In one embodiment, an insulin analog, according to the invention, may have increased solubility in relation to the precursor insulin. In an additional embodiment, an insulin analog, according to the invention, had increased solubility in relation to the precursor insulin at pH 3 to 9. Still in an additional embodiment, an insulin analog, according to the invention, had the increased solubility in relation to precursor insulin at pH 4 to 8.5. Still in an additional embodiment, an insulin analog, according to the invention, had increased solubility in relation to the precursor insulin at pH 4 to 8. Still in an additional embodiment, an insulin analog according to the invention had the solubility increased in relation to the precursor insulin at pH 4.5 to 8. Still in an additional modality, an insulin analogue, according to the invention, had the solubility increased in relation to the precursor insulin at pH 5 to 8. Still in a modality additionally, an insulin analogue according to the invention had an increased solubility in relation to the precursor insulin at pH 5.5 to 8. In an additional embodiment, an insulin analogue according to the invention had an increased solubility in relation to insulin precursor at pH 6 to 8.

In one embodiment, an insulin analog, according to the invention, had increased solubility in relation to the precursor insulin at pH 2 to 4.

By "increased solubility at a given pH it is understood that a higher concentration of an insulin analog of the invention dissolves in an aqueous solution or buffer at the pH of the solution, in relation to insulin is compared to, that is, human insulin or precursor insulin. Methods for determining whether the insulin contained in a solution is dissolved are known in the art.

In one embodiment, the solution can be centrifuged for 20 minutes at 30,000 g and then the concentration of insulin in the supernatant can be determined by RP-HPLC. If this concentration is equal, within the experimental error, to the concentration of insulin originally used to make the composition, then the insulin is completely soluble in the composition of the invention.

In another embodiment, the solubility of insulin in a composition of the invention can be simply determined by examining with the naked eye the container in which the composition is contained. Insulin is soluble if the solution is clear to the naked eye and no particulate matter is suspended or precipitated on the sides / bottom of the container.

An insulin analogue according to the invention may have increased potency and / or bioavailability in relation to the precursor insulin when compared by measurement.

Standard assays to measure the potency or bioavailability of insulin are known to the person skilled in the art and include (1) insulin radioreceptor assays, in which the relative potency of an insulin is defined as the ratio of insulin to analogue of insulin. insulin needed to displace 50% of the ¹²⁵ l-insulin specifically bound to the insulin receptors present on cell membranes, for example, a fraction of a rat liver plasma membrane; (2) lipogenesis assays performed, for example, with rat adipocytes, in which the relative potency of insulin is defined as the ratio of insulin to the insulin analog needed for maximum conversion of glucose [3- ³ H] into organic material it is extractable (that is, lipids); (3) glucose oxidation assays in isolated fat cells in which the relative potency of the insulin analog is defined as the ratio of insulin to the insulin analog to achieve 50% of the maximum glucose-1 conversion - [ ¹⁴ C] in ¹⁴ [CO ₂ j; (4) insulin radioimmunoassays that can determine the immunogenicity of insulin analogs by measuring the effectiveness by which insulin or an insulin analogue competes with ¹²⁵ l-insulin in binding specific anti-insulin antibodies; and (5) other assays that measure the binding of insulin or an insulin analogue to antibodies in blood plasma samples from animals, such as ELISA assays that have specific insulin antibodies.

Insulin analogs according to the invention can be optionally analyzed for additional protease sites that may be subject to additional substitutions of one or more hydrophobic amino acids with hydrophilic amino acids. An insulin analogue according to the invention can be an insulin analogue that has at least two hydrophilic acids at protease sites compared to the precursor insulin, the first modified insulin, and that still has at least one amino acid substitution. in a new protease site of the first modified insulin in which at least one hydrophobic amino acid has been replaced with at least one hydrophilic amino acid.

In one embodiment, an insulin analog is provided in which the B Terminal C chain is linked to the A Terminal N chain with 3 to 15 amino acids. Such an insulin analog is in this application denoted “single chain insulin or“ SCI ”and has the general structure BCA in which B is the human insulin B chain or an analogue or derivative thereof, A is the human insulin chain A or an analog or derivative and C is the peptide-binding chain of 3 to 15 amino acid residues normally binding B30 to A1. If the B chain is a desB30 chain, the binding peptide will join B29 with A1. Single-chain insulin will contain correctly positioned disulfide bridges (three) as in human insulin that are between CysA7 and CysB7 and between CysA20 and CysB19 and an internal disulfide bridge between CysA6 and CysA11. In one embodiment, an insulin analog is provided in which the B Terminal C chain is linked to the A Terminal N chain with 3 to 7 amino acids.

The present invention also relates to nucleic acid sequences that code for the claimed insulin analogs. In a further embodiment, the present invention relates to vectors that contain such nucleic acid sequences and host cells containing such nucleic acid sequences or vectors.

In a still further embodiment, the invention relates to a process for producing an insulin analog comprising (i) culturing a host cell comprising a nucleic acid sequence encoding an insulin precursor; (ii) isolating the insulin precursor from the culture medium and (iii) converting the insulin precursor to an insulin analog of the invention through an in vitro enzymatic conversion.

In a still further embodiment, the invention relates to a process for producing an insulin analog comprising (i) culturing a host cell comprising a nucleic acid sequence encoding an insulin precursor; (ii) isolating the insulin precursor from the culture medium and (iii) converting the insulin precursor into an insulin analog of the invention.

In one embodiment of the present invention, the host cell is a yeast host cell and, in an additional embodiment, the yeast host cell is selected from the genus Saccharomyces. In an additional modality, the yeast host cell is selected from Saccharomyces cerevisiae species.

By “insulin analog, as used in this application, is meant a polypeptide derived from the primary structure of a naturally occurring insulin, for example, that of human insulin, through the deletion and / or replacement of at least one amino acid residue that occurs in naturally occurring insulin and / or through the addition of at least one amino acid residue. The added and / or substituted amino acid residues can be encodable amino acid residues or other naturally occurring amino acid residues.

The insulin analogs according to the present invention can be human insulin or mutated analogs of it at one or more positions. In one embodiment, insulin analogs are designed to enhance stability against proteases based on the identified protease cleavage sites.

In one embodiment, at least one cleavage site subject to the mutation is in a position selected from the group consisting of: B2-3, B6-7, B9-10, B10-11, B13-14, B14-15, B16 -17, B22-23, B24-25, B2526, A13-14, A14-15 and A19-20 of human insulin.

In one embodiment, at least one cleavage site subject to the mutation is in a position selected from the group consisting of: B2-3, B6-7, B16-17, B22-23, B24-25 and / or A19- 20 of human insulin.

In one embodiment at least one cleavage site subject to the mutation is at position B22-23.

In one embodiment, at least one divination site subject to mutation is in a position selected from the group consisting of: B9-10, B10-11, B13-14, B14-15, B24-25, B25-26, A13 -14, A14-15.

In one embodiment, divage sites subject to mutation are in two or more positions selected from the group consisting of: B2-3, B6-7, B9-10, B10-11, B13-14, B14-15, B16- 17, B22-23, B24-25, B25-26, A13-14, A14-15 and A19-20 of human insulin.

In one embodiment, divation sites subject to mutation are in two or more positions selected from the group consisting of: B2-3, B6-7, B16-17, B22-23, B24-25 and / or A19-20 of human insulin.

In one embodiment, divage sites subject to mutation are in two or more positions selected from the group consisting of: B9-10, B10-11, B13-14, B14-15, B24-25, B25-26, A13- 14, A14-15.

In one embodiment, an insulin analogue according to the invention is obtained in which the A-chain of the insulin analogue comprises at least one mutation and the B-chain of the insulin analog comprises at least one mutation with respect to the precursor insulin, where at least one mutation in chain A is at one or more divination sites selected from the group consisting of: A13-14, A14-15 and A19-20 and at least one mutation in chain B is at one or more sites divage selected from the group consisting of: B2-3, B6-7, B9-10, ΒΙΟ11, B13-14, B14-15, B16-17, B22-23, B24-25 and B25-26.

In one embodiment, an insulin analog according to the invention is obtained in which the A-chain of the insulin analog comprises a mutation and the B-chain of the insulin analog comprises at least one mutation with respect to the precursor insulin, in which a mutation in chain A is at dividing site A19-20 and at least one mutation in chain B is at one or more dividing sites selected from the group consisting of: B2-3, B6-7, B16-17, B22-23 and B24-25.

In one embodiment, an insulin analog according to the invention is obtained in which the A chain of the insulin analog comprises at least one mutation and the B chain of the insulin analog comprises at least one mutation with respect to the precursor insulin, wherein at least one mutation in chain A is at one or more divination sites selected from the group consisting of: A13-14 and A14-15 and at least one mutation in chain B is at one or more divination sites selected from group consisting of: B9-10, B10-11, B13-14, B14-15, B24-25 and B25-26.

Amino acid residues suitable for substitution are selected with the aim of removing divage sites. In one embodiment, amino acids are selected with the additional goal of increasing solubility. In an additional embodiment, the increased solubility is in the pH range 3 to 9. Still in an additional embodiment, the increased solubility is in the pH range 4 to 8. The insulin or insulin analogue can be replaced in one or several positions any natural amino acid or any natural amino acid can be added to the precursor insulin or the precursor insulin analog. For convenience, here are the names of natural, codable amino acids, with the usual three letter codes & single letter codes in parentheses: Glycine (Gly & G), proline (Pro & P), alanine (Ala & A ), valine (Vai & V), leucine (Leu & L), isoleucine (lie & I), methionine (Met & M), cysteine (Cys & C), phenylalanine (Phe & F), tyrosine (Tyr & Y) , tryptophan (Trp & W), histidine (His & H), lysine (Lys & K), arginine (Arg & R), glutamine (Gin & Q), asparagine (Asn & N), glutamic acid (Glu & E) , aspartic acid (Asp & D), serine (Ser & S) and threonine (Thr & T). If, due to typing errors, there is a discrepancy with the commonly used codes, the commonly used codes apply. The amino acids present in the insulins of this invention are preferably amino acids that can be encoded by a nucleic acid. In one embodiment, insulin or an insulin analog is replaced by Gly, Glu, Asp, His, Gin, Asn, Ser, Thr, Lys, Arg and / or Pro and / or Gly, Glu, Asp, His, Gin, Asn , Ser, Thr, Lys, Arg and / or Pro is added to insulin or an insulin analogue. In one embodiment, insulin or an insulin analog is replaced by Glu, Asp, His, Gin, Asn, Lys and / or Arg and / or Glu, Asp, His, Gin,

Asn, Lys and / or Arg is added to insulin or an insulin analog.

The insulin analogs can be such that position 25 of the B chain can be modified from the natural residue Phe to His in combination with the substitution of the natural Tyr at position A14 for Glu, Asp or His. One or more additional mutations may include desB30 and the insulin analogs could be further modified via the extension of Terminal N or extension of Terminal C of chain A and / or chain B such as, for example, the extension of Terminal N of chain A and / or B of the insulin analog with GGP, GGPP, GP or GPP. Additional examples include, but are not limited to, additional mutations in which one or two Pro residues can be added to positions AO and / or B0, Leu can be added to position B31 and / or Glu can be added to position B32. One or more additional mutations can be selected from position A8 modified to His, position A21 to Gly, position B1 to Glu or Gin, position B16 to Glu, position B26 to Asp, position B27 to Glu and / or position B28 to Asp .

In one embodiment, an insulin analogue according to the invention is selected from the group consisting of:

human insulin A14E, B25H, desB30;

human insulin A14H, B25H, desB30;

human insulin A14E, B1E, B25H, desB30;

human insulin A14E, B16E, B25H, desB30;

human insulin A14E, B25H, B28D, desB30;

human insulin A14E, B25H, B27E, desB30;

human insulin A14E, B1E, B25H, B27E, desB30;

human insulin A14E, B1E, B16E, B25H, B27E, desB30;

human insulin A8H, A14E, B25H, desB30;

human insulin A8H, A14E, B25H, B27E, desB30;

human insulin A8H, A14E, B1E, B25H, desB30;

human insulin A8H, A14E, B1E, B25H, B27E, desB30;

human insulin A8H, A14E, B1E, B16E, B25H, B27E, desB30;

human insulin A8H, A14E, B16E, B25H, desB30;

human insulin A14E, B25H, B26D, desB30;

human insulin A14E, B1E, B27E, desB30;

human insulin A14E, B27E, desB30;

human insulin A14E, B28D, desB30;

human insulin AMD, B25H, desB30;

human insulin A (-1) P, A (0) P, A14E, B25H, desB30;

human insulin A14E, B (-1) P, B (0) P, B25H, desB30;

human insulin A (-1) P, A (0) P, A14E, B (-1) P, B (0) P, B25H, desB30;

human insulin A14E, B25H, B30T, B31L, B32E;

human insulin A14E, B25H;

human insulin A14E, B16H, B25H, desB30;

human insulin A14E, B10P, B25H, desB30;

human insulin A14E, B10E, B25H, desB30;

human insulin A14E, B4E, B25H, desB30;

human insulin A14H, B16H, B25H, desB30;

human insulin A14H, B10E, B25H, desB30;

human insulin A14E, B25H, desB27, desB28, desB29, desB30;

human insulin A13H, A14E, B10E, B25H, desB30;

human insulin A13H, A14E, B25H, desB30;

human insulin A14E, A18Q, B3Q, B25H, desB30;

human insulin A14E, B24H, B25H, desB30;

human insulin A8H, A14E, B10D, B25H, B26G, desB27, desB28, desB29, desB30;

human insulin A8H, A14E, B10D, B25H-amide, desB26, desB27, desB28, desB29, desB30;

human insulin A14E, B25H, desB26, desB27, desB28, desB29, desB30;

human insulin A14E, B25H, B26G, desB27, desB28, desB29, desB30;

human insulin A14E, B25H-amide, desB27, desB28, desB29, desB30 human insulin A14E, B25H, B26G, B27G, B28G, desB30; human insulin A14E, B26G, desB27, desB28, desB29, desB30;

human insulin A14E, B25H, desB26, desB27, desB28, desB29, desB30;

human insulin A14E, A18Q, A21Q, B3Q, B25H, desB30; human insulin A14E, A18Q, A21Q, B3Q, B25H, B27E, desB30;

human insulin A14E, A18Q, B3Q, B25H, desB30;

human insulin A13H, A14E, B1E, B25H, desB30;

human insulin A13N, A14E, B25H, desB30;

human insulin A13N, A14E, B1E, B25H, desB30;

human insulin A (-2) G, A (-1) P, A (0) P, A14E, B25H, desB30;

human insulin A14E, B (-2) G, B (-1) P, B (0) P, B25H, desB30;

human insulin A (-2) G, A (-1) P, A (0) P, A14E, B (-2) G, B (-1) P,

B (O) P, B25H, desB30;

human insulin A14E, B27R, B28D, B29K, desB30; human insulin A14E, B25H, B27R, B28D, B29K, desB30; human insulin A14E, B25H, B26T, B27R, B28D, B29K, desB30;

human insulin A14E, A18K, B25H, desB27, desB28, desB29, desB30;

human insulin A14E, A22K, B25H, desB27, desB28, desB29, desB30;

human insulin A14E, A22K, B25H, desB30;

human insulin A14E, desB1, desB2, desB3, B25H-amide, desB26, desB27, desB28, desB29, desB30;

human insulin A14E, desB1, desB2, desB3, B25H, desB27, desB28, desB29, desB30;

human insulin A14E, desB1, desB2, desB3, B16H, B25H, desB27, desB28, desB29, desB30;

human insulin A14E, B25H, B27R, desB30;

human insulin A14E, B25H, B27H, desB30; human insulin A14E, B25H, B27R, desB28-B30; human insulin A14E, B25H, B27H, desB28-B30; human insulin A14E, B25H, B27E, desB28-B30; human insulin A14E, B25H, B27K, desB28-B30; human insulin A14E, B27K, desB28-B30; human insulin A14E, A18Q, B3Q, B25H, desB30; human insulin A13E, A14E, B25H, desB30; human insulin A12E, A14E, B25H, desB30; human insulin A15E, A14E, B25H, desB30; human insulin A13E, B25H, desB30; human insulin A12E, B25H, desB30; human insulin A15E, B25H, desB30; human insulin A14E, B25H, desB27, desB30; human insulin A14E, B25H, B26D, B27E, desB30; human insulin EEAEAEAPK-B (1-29) -B25H-AAK-A (1-21) - A14E; human insulin EEAEPK-B (1-29) -B25H-DGK-A (1-21) -A14E; human insulin B (1-29) -B25H-AAK-A (1-21) -A14E; human insulin B (1-29) -B1E, B25H-AAK-A (1-21) -A14E; human insulin B (1-29) -B25H, B27E-AAK-A (1-21) -A8H A14E; human insulin B (1-29) -B1E, B25H, B27E-AAK-A (1-21) .A8H, A14E;

Human insulin EEAEAEAPK-B (1-29) -B16E, B25H-AAK-A (121) -A8H, A14E;

human insulin B (1-29) -B25H, B29Q-TGLGGGQ-A (1-21) -A14E

human insulin B (1-29) -B16E, B25H, B29Q-TGLGGGQ-A (1- 21) -A14E human insulin B (1-29) -B25H, B29Q-TGLGGGQ-A (1-21) -A8H, A14E human insulin A14E, B25H, B27R, desB30; human insulin A14E, B25H, B27N, desB30;

human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A13R, human insulin A13N, human insulin A13D, human insulin A13Q, human insulin A13E, human insulin A13G, human insulin A13H, human insulin A13K, human insulin A13S, human insulin A13T, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, human insulin A14E, AMR human insulin, A14N human insulin, AMD human insulin, A14Q human insulin, A14E human insulin, A14G human insulin, A14H human insulin,

B25H, B27D, desB30; B25H, B27Q, desB30; B25H, B27E, desB30; B25H, B27G, desB30; B25H, B27H, desB30; B25H, B27K, desB30; B25H, B27P, desB30; B25H, B27S, desB30; B25H, B27T, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; A14E, B25H, desB30; B16R, B25H, desB30; B16D, B25H, desB30; B16Q, B25H, desB30; B16E, B25H, desB30; B16H, B25H, desB30; B25H, desB30;

B25H, desB30;

B25H, desB30;

B25H, desB30;

B25H, desB30;

B25H, desB30;

B25H, desB30;

human insulin A8H, B10D, B25H; and human insulin A8H, A14E, B10E, B25H, desB30.

Insulin is a polypeptide hormone secreted by β cells in the pancreas. Insulin is composed of two polypeptide chains, A and B, which are linked by two interchain disulfide bridges. In addition, chain A has an intrachain disulfide bridge.

The hormone is synthesized as a single-chain precursor proinsulin (pre-proinsulin) composed of a 24-amino acid pre-peptide followed by a pro-insulin containing 86 amino acids in the configuration: pre-peptide-B-Arg Arg-C- Lys Arg-A, in which C is a 31 amino acid binding peptide. Arg-Arg and Lys-Arg are dividing sites for dividing the A and B chain binding peptide.

Three main methods were used to produce human insulin in microorganisms. Two involve Escherichia coli, with the expression of a large fusion protein in the cytoplasm (Frank et al (1981) in Peptides:.. Proceedings of the 7 ^th American Peptide Chemistry Symposium (Rich & Gross, eds), Pierce Chemical Co., Rockford, III. Pp 729739), or a signal peptide is used to allow secretion of the substance in the periplasmic space (Chan et al. (1981) PNAS 78: 5401-5404). A third method uses Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83: 6766-6770). The prior art discloses a number of insulin precursors that are expressed in E. coli or Saccharomyces cerevisiae, see US Patent No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

Insulin analogs are produced by expressing a DNA sequence that encodes the insulin analogue in question in a suitable host cell using the well-known technique as disclosed in, for example, US Patent No. 6500645. The insulin analogue it is expressed directly or as a precursor molecule that has a Terminal N extension in the B chain. This Terminal N extension can have the function of increasing the yield of the directly expressed product and can be up to 15 amino acid residues in length. The Terminal N extension must be cleaved in vitro after isolation from cell culture and therefore it will have a dividing site close to B1. N-terminal extensions of the appropriate type in the present invention are disclosed in US Patent No. 5,395,922 and European Patent No. 765,395A.

The isolated insulin analogue can be acylated in the desired position by well-known adlação methods and examples of such insulin analogues are described, for example, in European patent applications having the publication of ^Nos EP 214 826, EP 375 437 and EP 383472.

The nucleic acid sequence for encoding the respective polypeptide insulin analog can be prepared synthetically using established standard methods, for example, the phosphoramidite method described by Beaucage et al. (1981) Tetrahedron Letters 22: 1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3: 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and linked to form the synthetic DNA construct. A currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR).

The nucleic acid sequences can also be of genomic mixture, cDNA, and of synthetic origin. For example, a genomic sequence or cDNA that encodes a leader peptide can be combined with a genomic sequence or cDNA that encodes chains A and B, after which the DNA sequence can be modified at one site by inserting synthetic oligonucleotides that encode desired amino acid sequence for homologous recombination according to well-known procedures or preferably generate the desired sequence through PCR using suitable oligonucleotides.

The recombinant vector capable of replication in the selected microorganism or host cell and which carries a nucleic acid sequence encoding the polypeptide insulin analog in question can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, of which replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) to which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell's genome, or a transposon can be used. The vector can be linear or closed circular plasmids and preferably it will contain element (s) that allow the stable integration of the vector into the host cell genome or the autonomous replication of the vector in the genome independent cell.

In one embodiment, the recombinant expression vector is capable of replication in yeast. Examples of sequences that allow the vector to replicate in yeast are the REP 1-3 genes for replication and origin of replication in a 2 pm yeast plasmid.

Vectors can contain one or more selectable markers that allow easy selection of transformed cells. A selectable marker is the product of a gene that provides biocidal or viral resistance, resistance to heavy metals, prototrophic to auxotrophic, and the like. Examples of selectable bacterial markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as resistance to ampicillin, kanamycin, chloramphenicol or tetracycline. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyltransferase), pyrG (orotidine-5'-phosphate decarboxylase) and trpC (anthranylate synthase). Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A very suitable selectable marker for yeast is the TPI gene from Schizosaccharomyces pompe (Russell (1985) Gene 40: 125-130).

In the vector, the nucleic acid sequence is operably linked to a suitable promoter sequence. The promoter can be any nucleic acid sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding homologous or heterologous extracellular or intracellular polypeptides to the host cell.

Examples of promoters suitable for directing transcription in a bacterial host cell are promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase (dagA) gene, Bacillus subtilis levanasaccharase (sacB) gene, alpha-amylase gene Bacillus licheniformis (amyl), Bacillus stearothermophilus maltygenic amylase gene (amyM), Bacillus amyloliquefaciens (amyQ) alpha-amylase gene, and Bacillus licheniformis penicillinase (penP) gene. Examples of suitable promoters to direct transcription in a filamentous fungal host cell are promoters obtained from the Aspergillus oryzae TAKA amylase genes, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha amylase, and Aspergillus niger stable acid alpha-amylase. In a yeast host, useful promoters are the Saccharomyces cerevisiae Ma1, TPI, ADH or PGK promoters.

The nucleic acid construct will also typically be operationally linked to a suitable terminator. In yeast, a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1: 419434).

The procedures used to link the individual nucleic acid sequences contained in the expression vector, such as DNA encoding the desired polypeptide insulin analog, the promoter and the terminator, respectively, and inserting them into a suitable vector that contains the necessary information for replication in the selected host, they are well known to those skilled in the art. It will be understood that the vector can be constructed by the initial preparation of a DNA construct containing the entire DNA sequence encoding the insulin analog polypeptides of the invention, and subsequently inserting this fragment into a suitable expression vector, or successively through insertion of DNA fragments containing genetic information for the individual elements (such as signal, pro-peptide, binding peptide, A and B chains) followed by ligation.

The vector comprising such a nucleic acid sequence is introduced into the host cell so that the vector is maintained as an integral chromosomal part or as a self-replicating extrachromosomal vector as described above. The term host cell encompasses any progeny of a precursor cell that is not identical to the precursor cell due to mutations that occur during replication. The host cell can be a unicellular microorganism, for example, a prokaryote, or a non-unicellular microorganism, for example, a eukaryote. Useful single-cell cells are bacterial cells, such as gram-positive bacteria including, but not limited to, Bacillus cell, Streptomyces cell, or gram-negative bacteria, such as E. coli and Pseudomonas sp. Eukaryotic cells can be mammalian, insect, plant, or fungal cells. In a preferred embodiment, the host cell is a yeast cell. The yeast organism used in the process of the invention can be any suitable yeast organism that, in cultivation, produces large amounts of the inventive single-chain insulin. Examples of suitable yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia methida, Pichia, Candida, Pichia methidae, Pichia methida, Pichia methida, Pichia, pichia, pichia, pichia utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of yeast cells can, for example, be carried out by the formation of protoplasts followed by transformation in a manner known per se. The medium used for culturing the cells can be any conventional medium suitable for the growth of yeast organisms. The secreted polypeptide insulin analog, a significant proportion of which will be present in the medium in the properly processed form, can be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifuging, filtering or capturing the insulin precursor by a ion exchange matrix or a reverse phase absorption matrix, precipitation of the protein components of the supernatant or filtered through a salt, for example, ammonium sulfate, followed by purification through various chromatographic procedures, for example, exchange chromatography ion, affinity chromatography, or the like.

Pharmaceutical Compositions

Another objective of the present invention is to provide a pharmaceutical formulation comprising an insulin analogue according to the present invention that is present in a concentration of 0.1 mg / ml to 500 mg / ml, and wherein said formulation has a pH of 2.0 to 10.0. The formulation may further comprise protease inhibitor (s), a buffer system, preservative (s), tonicity agent (s), chelating agent (s), stabilizers and surfactants. In one embodiment of the invention, the pharmaceutical formulation is an aqueous formulation, that is, a formulation comprising water. Such a formulation is typically a solution or a suspension. In a further embodiment of the invention, the pharmaceutical formulation is an aqueous solution. The term aqueous formulation is defined as a formulation comprising at least 50% w / w of water. Likewise, the term aqueous solution is defined as a solution comprising at least 50% w / w of water, and the term aqueous suspension is defined as a suspension comprising at least 50% w / w of water.

In another embodiment, the pharmaceutical formulation is a vacuum frozen formulation, so that the doctor or the patient can add diluents and / or solvents before use.

In another embodiment, the pharmaceutical formulation is a dry formulation (for example, freeze-dried or spray-dried) ready for use without any prior dissolution.

In a further embodiment, the invention relates to a pharmaceutical formulation comprising an aqueous solution of an insulin analog of the present invention, and a buffer, wherein said insulin analog is present in a concentration of 0.1 mg / ml or above, and wherein said formulation has a pH of approximately 2.0 to approximately 10.0.

Formulations intended for oral use can be prepared according to any known method, and such formulations may contain one or more reagents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically tasty preparations. and palatable. Tablets can contain the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients that are suitable for the production of tablets. These excipients can be, for example, inert diluents, such as mannitol, maltodextrin, kaolin, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch; binding agents, for example, starch, gelatin, polymer or acacia; and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or may be coated by known techniques to delay the disintegration or release of the therapeutically active polypeptide.

The orally administrable formulations of this invention can be prepared and administered according to methods bemconhecidos in pharmaceutical chemistry, see Remington's Pharmaceutical Sciences, ^17th ed. (A. Osol ed., 1985).

In one embodiment of the invention, the pharmaceutical compositions of the present invention can be administered via solid dosage forms, such as tablets and capsules. The tablets can be prepared through wet granulation, through dry granulation, through direct compression or melt granulation.

Tablets of this invention can be prepared using conventional tablet pressing techniques. A general method of production involves mixing an insulin analog, a water-soluble diluent, a hydrophilic binder and optionally a portion of a disintegrant. This mixture is then granulated with an aqueous solution of the hydrophilic binder or an aqueous solution of the hydrophilic and surfactant binder and ground, if necessary. The granules are dried and reduced to an appropriate size. Any other ingredients, such as lubricants, (for example, magnesium stearate) and additional disintegrants, are added to the granules and mixed. This mixture is then compressed into a suitable size and shape using conventional tablet press machines, such as a rotary tablet press. The tablets can be film coated using techniques well known in the art.

Formulations for oral use can also be presented as hard or soft gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example, such as mannitol, maltodextrin, calcium carbonate, sodium carbonate, lactose, kaolin, phosphate calcium or sodium phosphate, or a soft gelatin capsule in which the active ingredient is mixed with water or an oily medium, for example, peanut oil, liquid paraffin, or olive oil.

Capsules for this invention can be prepared using conventional methods. A general method for production involves mixing a therapeutically active peptide, alginate, a water-soluble diluent, a hydrophilic binder, and optionally a portion of a disintegrant. This mixture is then granulated with an aqueous solution of the hydrophilic binder or an aqueous solution of the hydrophilic binder and surfactant in water, and ground, if necessary. The granules are dried and reduced to an appropriate size. Any other ingredients, such as a lubricant, are added to the granules and mixed. The resulting mixture is then filled into an appropriately sized hard gelatin capsule using conventional capsule filling machines.

In a further embodiment of the invention, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris (hydroxymethyl) 43 aminomethane, bicin, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers is an alternative embodiment of the invention.

In a further embodiment of the invention, the formulation further comprises a pharmaceutically acceptable preservative. The preservative is present in an amount sufficient to obtain a preservative effect. The amount of the preservative in a pharmaceutical formulation is well known to the skilled person and can be determined, for example, from literature in the field and / or known quantity (s) of preservatives, for example, in commercial products . Each of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th Edition, 1995.

In a further embodiment of the invention, the formulation further comprises a chelating agent. The use of a chelating agent in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th Edition, 1995.

In a further embodiment of the invention, the formulation further comprises a stabilizer. The term stabilizer as used in this application refers to chemicals added to pharmaceutical formulations containing polypeptide to stabilize the peptide, that is, to increase the shelf life and / or time in use of such formulations. The use of a stabilizer in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th Edition, 1995.

In a further embodiment of the invention, the formulation further comprises a surfactant. The term "surfactant" as used in this application refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, the head, and a fat-soluble (lipophilic) segment. Surfactants accumulate preferentially at interfaces, in which the hydrophilic part is oriented towards water (hydrophilic phase) and the lipophilic part towards oil - or hydrophobic phase (ie glass, air, oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micellar concentration or CMC. In addition, surfactants reduce the surface tension of a liquid. Surfactants are also known as antipathetic compounds. The term Detergent is a synonym used for surfactants in general. The use of a surfactant in pharmaceutical compositions is well known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th Edition, 1995.

In a further embodiment of the invention, the formulation further comprises protease inhibitors.

It is possible that other ingredients may be present in the pharmaceutical formulation of the insulin analog of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (for example, human serum albumin, gelatin or protein) and a zwitterion (for example, amino acid , such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing an insulin analog according to the present invention can be administered to a patient in need of such treatment at various sites, for example, at topical sites, for example, skin and mucous sites, at sites for absorption bypass, for example, administration in an artery, in a vein, in the heart, and in sites that involve absorption, for example, administration in the skin, subcutaneous, in a muscle or in the abdomen.

The administration of pharmaceutical compositions according to the invention can be by various routes of administration, for example, tingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or combination of them, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, urethral, and parenteral to patients in need of such treatment.

Compositions of the current invention can be administered in various dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, ointments, pastes, bandages, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatin capsules and soft capsules of ge10 latina, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalers, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transformation solutions, for example, in situ coagulation, in situ placement, in situ precipitation, crystallization in situ, infusion solution, and implants.

Compositions of the invention can be further combined in, or attached to, for example, through covalent, hydrophobic and electrostatic interactions, a drug vehicle, drug delivery system and advanced drug delivery system in order to further enhance this stability insulin analogue compound, increase bioavailability, increase solubility, reduce adverse effects, perform well-known chronotherapy for those skilled in the art, and increase patient adaptation or any combination thereof.

Compositions of the present invention are useful in the formulation of solids, semi-solids, powders and solutions for pulmonary administration of the insulin analogue, using, for example, a metered dose inhaler, dry powder inhaler and nebulizer, all of which are devices well known to the skilled in the art.

Compositions of the current invention can be useful in formulating controlled, sustained, prolonged, delayed, and slow drug delivery systems. More specifically, but not limited to, the compositions can be useful in the formulation of controlled parenteral release and sustained delivery systems (both systems lead to an often reduced number of administrations), well known to those skilled in the art. Even more preferably, they are controlled-release and sustained-release systems administered subcutaneously. Without limiting the scope of the invention, examples of useful controlled-release systems and compositions are hydrogels, oil gels, liquid crystals, polymeric micelles, microspheres, nanoparticles.

Methods for producing controlled release systems useful for compositions of the present invention include, but are not limited to, crystallization, condensation, cocrystallization, precipitation, coprecipitation, emulsification, dispersion, high pressure homogenization, encapsulation, spray drying, microencapsulation, coacervation , phase separation, solvent evaporation for the production of microspheres, extrusion and supercritical fluid processes. General reference is made to the Handbook of Pharmaceutical Controlled Release (Wise, D.L., ed. Marcei Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, E.J., ed. Marcei Dekker, New York, 2000).

Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection using a syringe, optionally a pen-type syringe. Alternatively, parenteral administration can be performed using an infusion pump. An additional option is a composition that can be a solution or suspension for the administration of the insulin analog compound in the form of a nasal or pulmonary spray. As an additional option, pharmaceutical compositions containing the insulin analog compound of the invention can also be adapted for transdermal administration, for example, by needle-free injection or by an adhesive, optionally an iontophoretic, or transmucosal, for example, oral administration .

The insulin analogue according to the invention can be administered via the pulmonary route in a vehicle, as a solution, suspension or dry powder using any of the known types of devices suitable for pulmonary drug delivery. Examples of these comprise, but are not limited to, the three general types of aerosol generation for the delivery of pulmonary drug, and may include jet or ultrasonic nebulizers, metered dose inhalers, or dry powder inhalers (Cf. Yu J , Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14 (4) (1997) 395-453).

In an additional embodiment, the formulation can be aerosolized by some known aerosolization technology, such as nebulization, to achieve an MMAD of aerosol particles less than 10 pm, more preferably between 1 to 5pm, and even more preferably between 1 to 3 pm. The preferred particle size is based on the most effective size for delivering the drug to the deep lung, where the protein is optimally absorbed (cf. Edwards DA, Ben-Jebria A, Langer A, Recent advances in pulmonary drug delivery using large, porous inhaled particles J Appl Physiol 84 (2) (1998) 379-385).

Deposition in the deep lung of pulmonary formulations comprising the insulin analog compound can optionally be further optimized through the use of modifications of inhalation techniques, for example, but not limited to: slow inhalation flow (for example, 30 L / min), breath retention and activation time.

The term stabilized formulation refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.

The term physical stability of the protein formulation as used in this application refers to the protein's tendency to form biologically inactive and / or insoluble aggregates of the protein as a result of exposure of the protein to thermomechanical stress and / or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. The physical stability of aqueous protein formulations is assessed by visual inspection and / or turbidity measures after exposure of the filled formulation in suitable containers (eg cartridges or bottles) to mechanical / physical stress (eg agitation) at 48 different durations at various time periods. Visual inspection of the formulations is carried out in a spot light focused on a dark background. The turbidity of the formulation is characterized by a visual note that positions the degree of turbidity, for example, on a scale of 0 to 3 (a formulation showing that no turbidity is equivalent to a visual score 0, and a formulation showing that the visual turbidity in the daylight is equivalent to visual note 3). A formulation is classified as physically unstable with respect to protein aggregation, when it shows visual turbidity in daylight. Alternatively, the turbidity of the formulation can be assessed by simple turbidity measurements well known to the skilled person. The physical stability of aqueous protein formulations can also be assessed through the use of a spectroscopic agent or probe for the conformational position of the protein. The probe is preferably a small molecule that preferably binds to a non-native protein conformer. An example of a small molecular spectroscopic probe of the protein structure is Tioflavin T. Tioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and possibly other protein configurations as well, Tioflavin T gives rise to a new excitation maximum at approximately 450nm and potentiated emission at approximately 482nm when bound to a form of fibril protein. Unbound Tioflavin T is essentially non-fluorescent at wavelengths.

Other small molecules can be used as probes for changes in the natural protein structure to non-native states. For example, “hydrophobic part” probes that preferentially bind to exposed hydrophobic parts of a protein. Hydrophobic parts are usually buried in the tertiary structure of a protein in its native state, but become exposed as a protein begins to open or denature itself. Examples of these small molecular spectroscopic probes are aromatic, hydrophobic dyes, such as anthracene, acridine, phenanthroline or the like. Other spectroscopic probes are metal amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.

The term chemical stability of the protein formulation as used in this application refers to covalent chemical modifications in the protein structure leading to the formation of chemical breakdown products with possible lower biological potency and / or increased potential immunogenic properties compared to the native protein structure. Various chemical breakdown products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation most likely cannot be completely avoided and increased amounts of chemical degradation products are often seen during storage and use of the protein formulation, as is well known to the person skilled in the art. Most proteins are prone to deamidation, a process in which the side chain amide group in the glutaminyl or asparaginyl residues is hydrolyzed to form a free carboxylic acid. Other degradation pathways involve the formation of high molecular weight transformation products where two or more protein molecules are covalently linked to each other by transamidation and / or disulfide interactions leading to the formation of linked dimer, oligomer and polymer degradation products covalently, (Stability of Protein Pharmaceuticals, Ahem. TJ & Manning MC, Plenum Press, New York 1992). Oxidation (from, for example, methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein formulation can be assessed by measuring the amount of chemical degradation products at various points in time after exposure to different environmental conditions (the formation of degradation products can often be accelerated, for example, by increasing the temperature). The amount of each individual degradation product is often determined by separating the degradation products depending on the charge and / or size of the molecule using various chromatography techniques (for example, SEC-HPLC and / or RP-HPLC).

Therefore, as outlined above, a "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a formulation must be stable during use and storage (according to the recommended conditions of use and storage) until the expiration date is reached.

In one embodiment of the invention the pharmaceutical formulation comprising the insulin analog compound is stable for more than 6 weeks of use and for more than 3 years of storage.

In another embodiment of the invention the pharmaceutical formulation comprising the insulin analog compound is stable for more than 4 weeks of use and for more than 3 years of storage.

In a further embodiment of the invention, the pharmaceutical formulation comprising the insulin analog compound is stable for more than 4 weeks of use and for more than two years of storage.

In yet a further embodiment of the invention the pharmaceutical formulation comprising the insulin analog compound is stable for more than two weeks of use and for more than two years of storage.

Aqueous suspensions can contain the active compounds in the mixture with excipients suitable for the production of aqueous suspensions.

Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, for example, peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil, such as liquid paraffin. Oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those presented above, and flavoring agents can be added to provide a palatable oral preparation. These formulations can be preserved by adding an antioxidant, such as ascorbic acid.

Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the active compound in the mixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by that already mentioned above. Additional excipients, for example, sweetening, flavoring, and coloring agents, may also be present.

Pharmaceutical formulations comprising a compound for use according to the present invention can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, for example, olive oil or peanut oil, or a mineral oil, for example, a liquid paraffin, or a mixture thereof. Suitable emulsifying agents can be naturally occurring gums, for example, acacia or tragacanth gum, naturally occurring phosphatides, for example, soybeans, lecithin, and partial esters or esters obtained from anhydride fatty acids and hexitol, for example, mono- sorbitan oleate, and condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. Emulsions can also contain sweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations can also contain a soothing, preservative and flavoring and colorifying agent.

In a further embodiment of the invention, the formulation further comprises a permeation enhancer. It is often considered that bile salts and fatty acids increase the oral permeability of lipid bilayer membranes of the epithelial cell lining of the GI tract. In general, permeation enhancers increase the paracellular and transcellular transport of macromolecules by reversibly altering the integrity of the membrane. The bile salt is selected from the group consisting of cholate, deoxycholate, taurocholate, glycocholate, taurodeoxycholate, ursodeoxycholate, tauroursodeoxycholate, and quenodeoxycholate. Fatty acids are selected from the group of short, medium and long chain fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, etc. Other enhancers can be surfactants, such as monoglycerides, polyoxyethylene esters, sorbitan surfactants (non-ionic) and sulfates (anionic).

In a further embodiment of the invention, the formulation further comprises a mucoadhesive polymer. An intimate contact of the drug delivery system with the mucosa of the gastrointestinal tract can be obtained through the use of such mucoadhesive polymer. Close contact of the dosage form with the membrane appears advantageous since an enzymatic degradation of the therapeutically active polypeptide en route between the delivery system and the absorbent membrane can be avoided. In addition, a concentration gradient step on the absorbent membrane representing the driving force for passive drug absorption can be provided.

In a further embodiment of the invention, the formulation further comprises a proteolytic enzyme (s) inhibitor (s) to bypass, in addition, the enzymatic barrier and carrying out the release of the therapeutically active polypeptide, such as aminopeptidase inhibitor, amastatin, bestatin, boroleucine and puromycin. Examples of protease inhibitors are sodium glycolate, camostate mesylate, bacitracin, soybean trypsin inhibitor and aprotinin.

Microencapsulation and encapsulation are techniques used in drug delivery systems for therapeutically active polypeptides to optimize release properties including protection from enzymatic degradation. Microencapsulation or encapsulation can be in the form of polymeric drug delivery systems, such as hydrogels and nanocapsules / microspheres, and lipid drug delivery systems, such as liposomes and microemulsions.

Formulations of the present invention can be administered in various dosage forms, for example, as solutions, suspensions, micro- and nanosuspension, emulsions, microemulsions, multiple emulsion, foams, ointments, pastes, ointments, tablets, coated tablets, effervescent tablets, sublingual tablets, oral tablets, capsules, for example, hard gelatin capsules and soft gelatin capsules, powder, granules, in situ processing solutions, for example, in situ gelation, in situ solidification, in situ precipitation, in crystallization in situ, stomach flotation formulation, such as flotation suspension, flotation tablet or the like.

In another embodiment, the present invention relates to an insulin analog according to the invention for use as a medicament.

In one embodiment, an insulin analogue according to the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyspilidemia, cognitive disorders, atherosclerosis, myocardial infarction, stroke, coronary heart disease and other cardiovascular disorders, inflammatory bowel syndrome, dyspepsia and gastric ulcers.

In another embodiment, an insulin analogue according to the invention is used as a drug to slow or prevent disease progression in type 2 diabetes.

In another embodiment, an insulin analogue according to the invention is used as a medicine for reducing food intake, reducing apoptosis of β cells, increasing the function of β cells and the number of β cells and / or for restoring sensitivity β cells to glucose.

In one embodiment of the invention, the derivative according to the invention is for use as a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyspilidemia, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers or to slow or prevent the progression of the disease in type 2 diabetes or to reduce food intake, reduced apoptosis of β cells, increased β cell function and the number of β cells and / or for restoring the sensitivity of β cells to glucose, is provided.

In a further embodiment of the invention, a method for treating or preventing hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyspilidemia, cognitive disorders, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers or to slow or prevent disease progression in type 2 diabetes or to reduce food intake, reduce β cell apoptosis, increase β cell function and amount of β cells and / or for restoring the sensitivity of β cells to glucose, the method comprising administering to a patient in need of such treatment an effective amount of such treatment of an insulin analog according to the invention, is provided.

Treatment with an insulin analogue according to the present invention can also be combined with a second or more pharmacologically active substances, for example, selected from antidiabetic agents, anti-obesity agents, appetite regulating agents, antihypertensive agents, agents for treating and / or preventing complications resulting from or associated with diabetes and agents for treating and / or preventing complications and disorders resulting from or associated with obesity. Examples of these pharmacologically active substances are: GLP-1 and GLP-1 derivatives and analogs, GLP-2 and GLP-2 derivatives and analogs, Exendina-4 and Exendin-4 derivatives and analogs, amylin and amylin derivatives and analogs, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, liver enzyme inhibitors involved in the stimulation of gluconeogenesis and / or glycogenolysis, modulators of glucose absorption, compounds that modify glucose lipid metabolism, such as anti-hyperlipidemic people such as HMG-CoA inhibitors (statins), compounds that reduce food intake, RXR agonists and agents that act on the ATP-dependent potassium channel of β cells; Coles55 tyramine, colestipol, clofibrate, genfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; β-blockers, such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors, such as benazepril, captoprila, enalaprila, fosinoprila, lisinoprila, alatrioprila, quinaprila and canalami calcium, such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamila, and β-blockers, such as doxazosin, urapidyl, prazosin and terazosin; CART agonists (cocaine and amphetamine regulated transcript), NPY antagonists (neuropeptide Y), MC4 agonists (melanocortin 4), orexin antagonists, TNF (tumor necrosis factor) agonists, CRF agonists (CRF release factor) corticotropin), CRF BP antagonists (corticotropin releasing factor binding protein), urocortin agonists, β3 agonists, MSH (melanocyte stimulating hormone) agonists, MCH (melanin concentrating hormone) antagonists, CCK ( cholecystokinin), serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT agonists (serotonin), bombesin agonists, galanine antagonists, growth hormone, growth hormone release compounds , TRH agonists (thyrotropin releasing hormone), UCP 2 or 3 modulators (uncoupling protein 2 or 3), leptin agonists, agonists of DA (bromocriptine, doprexin), lipase / amylase inhibitors, RXR (retinoid X receptor) modulators, TR β agonists; histamine H3 antagonists, gastrin and gastrin analogues and derivatives.

It should be understood that any suitable combination of the derivatives according to the invention with one or more of the aforementioned compounds and optionally one or more pharmacologically active substances is within the scope of the present invention.

All references, including publications, patent applications, and patents, cited in this application are hereby incorporated by reference in their entirety and to the same extent, as if each reference were individually and specifically indicated to be incorporated by reference and were presented in its entirety in this order (up to the maximum limit permitted by law).

All titles and subtitles are used in this application for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (for example, "as") provided in this application, is intended simply to further clarify the invention and is not a limitation on the scope of the invention unless otherwise claimed. No language in the report should be interpreted as indicating any element not claimed as essential to the practice of the invention.

The citation and incorporation of patent documents in this application is for convenience only and does not reflect any view of the validity, patentability and / or applicability of such patent documents.

This invention includes all modifications and equivalents of the subjects referred to in the attached claims as permitted by applicable law.

EXAMPLES

EXAMPLE 1

Comparison of proteolytic stability (half-life) of B25H, A14E,

A14E B25H and human insulin in relation to pepsin, chymotrypsin, and carboxypeptidase A

The proteolytic stability of human insulin and insulin analogs (0.06 mM, 10 pL) in relation to chymotrypsin or pepsin (0.34 to 3.4 pg) was measured after incubation in 100 mM NH ₄ HCO3a, pH 8 , 1 or 10 mM HCI, pH 2.0, respectively and 25 ° C in a final volume of 100 pL. At various times (0, 5, 15, 30 and 60 minutes) the samples were quenched with an equal volume of 0.5% TFA or 0.1 m TrisHCI, pH 8.0 (final pH 7.7) and transferred to 5 ° C. Human insulin and insulin analogs were immediately analyzed by RP-HPLC at 214 nm and the area under the peak corresponding to the intact protein was determined.

The stability of human insulin and insulin analogs (0.06 mM, 13.9 pg) in relation to carboxypeptidase A (6.8 pL of the working solution, 20 mg / ml, 53 units / mg, Sigma C-9268) it was measured after incubation in 5 mM NaP, 140 mM NaCI, 70 ppm Tween20, pH 7.4 and 37 ° C in a final volume of 400 pL. At various times (5, 15, 30, 60 minutes) the samples were quenched with an equal volume of 0.2% TFA and transferred to 5 ° C. The reference samples (0 minutes) were prepared without adding the enzymes. Human insulin and analogs were immediately analyzed by RP-HPLC in a 214 nm neutral buffer system and the area under the peak corresponding to the intact protein was determined. Similar retention times have been observed for human insulin and desB30. half-lives (Ti / ₂ ) were obtained from the curves and the increase / decrease compared to human insulin was calculated (relative stability), similar half-lives were determined for human insulin, Tv ₂ =

6.9 minutes, and desB30 human insulin , Ti / ₂ - 6.7. Mutation Site (s) in Human Insulin T _1/2 [Min] (Times) Pepsin T _1/2 (Min) / Times Chymitrypsin T _1/2 (Min) / Times Carboxypeptidase A B25H 17.8 (16.2) 25.7 (5.1) 25.7 (2.1) none 1.1 (1.0) 5.0 (1.0) 6.9 (1.0) A14E, DESB30 3.7 (3.4) 33.2 (6.6) 15.7 (2.3) none 1.1 (1.0) 5.0 (1.0) 6.9 0.0) A14E, B25H, DESB30 42.4 (38.5) 62.4 (12.5) 61.9 (9.0) none 1.1 (1.0) 5.0 (1.0) 6.9 (1.0)

EXAMPLE 2

Identification of protease dividing sites in insulin by MS analysis and enzyme specificity

The identification of protease divination sites was performed 20 by the time course analysis of limited insulin proteolysis and insulin analogs by various enzymes (pepsin, chymotrypsin, carboxypeptidase A) by LC-MS. The peptide fragments generated were identified by MS or MS / MS and quantified at 214 nm in order to identify major and minor dividing sites (hot spots). The following hot spots of pepsin (B24-25, B25-26, B26-27) and chymotrypsin (B16-17, B24-25, B25-26, B26-27, A14-15 and A19-20) within human insulin have been identified. Interestingly, the hot spots are overlaid with the insulin-degrading enzyme (IDE), seen below, and cathepsin D (Hanne Refsgaard, Novo Nordisk A / S, personal communication).

The divination sites in the insulin sequence were also predicted based on the enzymatic specificity of chymotrypsin, trypsin and insulin degrading enzyme (IDE) as shown in example 3 below. The divination sites predicted for trypsin (basic residues, C-term for Lys, Arg), elastase (aliphatic residues, C-term for Ala, Vai, Gly, Ser), carboxypeptidase A (broad specificity, but not for Arg , Lys & Pro), pepsin A (aromatic residues, N-term for Trp, Tyr, Phe, and Leu and several other residues), and chymotrypsin (aromatic residues, C-term for Trp, Tyr, Phe, and Leu , but not Xxx-Pro) are described in several biochemistry manuals. Insulin IDE dividing sites were recently published: B9-10, ΒΙΟ11, B13-14, B14-15, B24-25, B25-26, A13-14, A14-15 (Duckworth et al., 2006) and showed the following substitutions are said to inhibit IDE degradation of insulin: B10D and B4E_B16Q_B17F (Bennett et al., 2003). EXAMPLE 3

Repeated cycles of identifying hot spots in human insulin

A14E, B25H, desB30

The main chymotrypsin dividing hot spots (B16-17, B24-25, A19-20) and minor dividing sites (B1-2, B6-7) were identified in human insulin A14E, B25H, desB30 according to the method described in example 2. Insulin analogues were designed based on the identified divination sites and predicted divage sites for enzyme specificity. Amino acid residues for substitution were selected in order to remove divage sites and increase solubility.

Chymotrypsin, analogue design based on hot spots identified by LC-MS of human insulin digests A14E, B25H, desB30:

Α14Χ1 Β25Η desB30X12 ΟΙ B1X2 B6X3 B16X6 B24X8 A19X11

Χ1 = E / D / H / Q / N / S / T / M / P

Χ2 = Native E / D / H / Q / N / S / T / M / P / aa

X3 = Native E / D / H / Q / N / S / T / M / P / aa

X6 = Native E / D / H / Q / N / S / T / M / P / aa

X8 = Native E / D / H / Q / N / S / T / M / P / aa

X11 = Native E / D / H / Q / N / S / T / M / P / aa

X12 = native desB30 / aa

Note: replacing B24e / or A19 in human insulin is likely to significantly reduce affinity for the receptor.

A14X1 B25P desB30X12 ΟΙ B1X2 B6X3 B16X6 B24X8 A19X11

XI = E / D / H / Q / N / S / T / M / P

X2 = Native E / D / H / Q / N / S / T / M / P / yy X3 = Native E / D / H / Q / N / S / T / M / P / yy X6 = E / D / Native H / Q / N / S / T / M / P / aa X8 = Native E / D / H / Q / N / S / T / M / P / aa

XII = E / D / H / Q / N / S / T / M / P / aa native X12 = desB30 / aa native

Please note: replacing B25 with Pro is likely to significantly reduce the chymotrypsin breakdown in B24-25.

Chymotrypsin, project based on identified and predicted hot spots:

A14X1 B25H desB30X12 ΟΙ B1X2 B6X3 B11X4 B15X5 B16X6 B17X7 B24X8 A13X9 A16X10 A19X11

X1 = E / D / H / Q / N / S / T / M / P X2 = E / D / H / Q / N / S / T / M / P / native X3 = E / D / H / Q / N / S / T / M / P / yy native X4 = E / D / H / Q / N / S / T / M / P / yy native X5 = E / D / H / Q / N / S / T / M / P / yy native X6 = E / D / H / Q / N / S / T / M / P / yy native X7 = E / D / H / Q / N / S / T / M / P / yy native X8 = E / D / H / Q / N / S / T / M / P / aa native

Χ9 = Native E / D / H / Q / N / S / T / M / P / aa

X10 = Native E / D / H / Q / N / S / T / M / P / aa

X11 = Native E / D / H / Q / N / S / T / M / P / aa

X12 = native desB30 / aa

Note: replacing B11, B15, B24, A13, A16, and / or A19 in human insulin is likely to significantly reduce affinity for the receptor.

Chymotrypsin + trypsin, project based on identified and predicted hot spots:

A14X1 B25H desB30X12 ΟΙ B1X2 B6X3 B11X4 B15X5 B16X6 B17X7 B24X8 A13X9 A16X10 A19X11 B22X13

XI = E / D / H / Q / N / S / T / M / P

X2 = Native E / D / H / Q / N / S / T / M / P / aa

X3 = Native E / D / H / Q / N / S / T / M / P / aa

X4 = Native E / D / H / Q / N / S / T / M / P / aa

X5 = Native E / D / H / Q / N / S / T / M / P / aa

X6 = Native E / D / H / Q / N / S / T / M / P / aa

X7 = Native E / D / H / Q / N / S / T / M / P / aa

X8 = Native E / D / H / Q / N / S / T / M / P / aa

X9 = Native E / D / H / Q / N / S / T / M / P / aa

X10 = Native E / D / H / Q / N / S / T / M / P / aa

XII = Native E / D / H / Q / N / S / T / M / P / aa

X12 = native desB30 / aa

X13 = Native E / D / H / Q / N / S / T / M / P / aa

Chymotrypsin + trypsin + insulin degradation enzyme (IDE), project based on identified and predicted hot spots:

A14X1 B25H desB30X12 ΟΙ B1X2 B6X3 B11X4 B15X5 B16X6 B17X7 B24X8 A13X9 A16X10 A19X11 B22X13 B4X14 B9X15 B10X16 B13X17 B14X18

X1 = E / D / H / Q / N / S / T / M / P

X2 = Native E / D / H / Q / N / S / T / M / P / aa

X3 = Native E / D / H / Q / N / S / T / M / P / aa

Χ4 = Native E / D / H / Q / N / S / T / M / P / aa

X5 = Native E / D / H / Q / N / S / T / M / P / aa

X6 = Native E / D / H / Q / N / S / T / M / P / aa

X7 = Native E / D / H / Q / N / S / T / M / P / aa

X8 = Native E / D / H / Q / N / S / T / M / P / aa

X9 = Native E / D / H / Q / N / S / T / M / P / aa

X10 = Native E / D / H / Q / N / S / T / M / P / aa

X11 = Native E / D / H / Q / N / S / T / M / P / aa

X12 = native desB30 / aa

X13 = Native E / D / H / Q / N / S / T / M / P / aa

X14 = Native E / D / H / Q / N / S / T / M / P / aa

X15 = Native E / D / H / Q / N / S / T / M / P / aa

X16 = Native E / D / H / Q / N / S / T / M / P / aa

X17 = Native E / D / H / Q / N / S / T / M / P / aa

X18 = E / D / H / Q / N / S / T / M / P / aa native EXAMPLE 4

Proteolytic stability (half-life) of insulin analogues in relation to chymotrypsin

Insulin analogs were tested in stability assays and analogs exhibiting enhanced resistance to proteolytic digestion were identified through increased half-lives according to the methods described in example 1. The results demonstrate the potential of the improved insulin analogs in the increased bioavailability due to potentiated resistance to proteolytic degradation and improved solubility.

The stability of the following insulin analogs with respect to chymotrypsin has been tested against human insulin:

Insulin analogues Digestion by Chimo- trypsin [Times of stability in relation to human insulin] human sulina A14E, B25H, desB27, desB28, desB29, desB30 43.8 human sulina A8H, A14E, B25H, B27E, desB30 38.9 human sulin EEAEAEAPK-B (1-29) -B25H-AAK-A (1-21) - A14E 33.2 human sulina A8H, A14E, B1E, B25H, B27E, desB30 22.8 human sulina A14E, B25H, B28D, desB30 17.4 human sulina A14E, B25H, B26D, desB30 16.6 human sulina A14E, B25H, B27E, desB30 14.1 human sulina A8H, A14E, B25H, desB30 13.3 human sulina A8H, A14E, B10E, B25H, desB30 12.6 human sulina A14E, B25H, desB30 12.5 human sulina A14E, B1E, B25H, B27E, desB30 11.9 human sulina A8H, A14E, B1E, B25H, desB30 10.2 human sulina A8H, A14E, B1E, B16E, B25H, B27E, desB30 8.3 human sulina A14D, B25H, desB30 7.9 human sulina A14E, B27E, desB30 7.7 human sulina A14E, B28D, desB30 7.5 human sulina A8H, B10D, B25H 7.4 human sulina A14E, B1E, B16E, B25H, B27E, desB30 5.5 human sulina A14E, B1E, B25H, desB30 4.8 human sulina A14H, B25H, desB30 4.5 human sulina A14E, B25H 4.1 human sulina A14E, B1E, B27E, desB30 3.9 human sulina A8H, A14E, B16E, B25H, desB30 2.3 human sulina A14E, B16E, B25H, desB30 2.1 human sulina (reference) 1

EXAMPLE 5

Proteolytic stability of insulin analogues in relation to rat intestine extracts (duodenum lumen)

Insulin analogues were tested for stability against proteolytic degradation in intestinal extracts. The extracts were prepared by washing the luminal content of fasting animals. Insulin analogues were incubated with extracts diluted at 37 ° C at pH = 7.4 and after 20 hours of incubation the concentration of the entire compound was determined by RP-HPLC. The relative amounts of whole insulin analogs and intact human insulin were compared. Approximately 5 to 10-fold difference in stability between human insulin and human insulin analog A14E B25H desB30 was found.

Sequence Listing <110> NEW NouDISK A / S <120> Protease resistant insulin analogs <130> 7484.204-WO <160> 4 <170> Patent version 3.4 <210> 1 <211> 25 <212> PRT <213 > Artificial <220>

<223> Modified Insulin A chain <220>

<221> MISC_FEATURE <222> (1) .. (1) <223> Xaa is absent or Gly <220>

<221> MISC_FEATURE <222> (2) .. (2) <223> Xaa is absent or Pro <220>

<221> MISCFEATURE <222> (3) .. (3) <223> Xaa is absent or Pro <220>

<221> MISC FEATURE <222> (11) .. (11) <223> Xaa is Thr or His <220>

<221> MISC FEATURE <222> (15) .. (15) <223> Xaa is Ser, Asp or Glu <220>

<221> MISC FEATURE <222> (16) .. (16) <223> Xaa is Leu, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser or Glu <220>

<221> MISC FEATURE <222> (17) .. (17) <223> Xaa is Tyr, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser or Glu <220>

<221> MISC FEATURE <222> (18) .. (18) <223> Xaa is Gin, Asp or Glu <220>

<221> MISC_FEATURE <222> (21) .. (21) <223> Xaa is Asn, Lys or Gin <220>

<221> MISC FEATURE <222> (24) .. (24) <223> Xaa is Asn or Gin <220>

<221> MISC_FEATURE <222> (25) .. (25) <223> Xaa is absent or Lys <400> 1

Xaa Xaa Xaa Gly lie Vai Glu Gin Cys Cys Xaa Ser lie Cys Xaa Xaa 15 10 15

Xaa Xaa Leu Glu Xaa Tyr Cys Xaa Xaa

25 <210> 2 <211> 35 <212> PRT <213> Artificial <220>

<223> Modified Insulin B chain <220>

<221> MISCFEATURE <222> (1) .. (1) <223> Xaa is absent or Gly <220>

<221> MISC FEATURE <222> (2) .. (2) <223> Xaa is absent or Pro <220>

<221> MISC FEATURE <222> (3) .. (3) <223> Xaa is absent or Pro <220>

<221> MISC FEATURE <222> (4) .. (4) <223> Xaa is absent Phe or Glu <220>

<221> MISC_FEATURE <222> (5) .. (5) <223> Xaa is absent or Vai <220>

<221> MISCFEATURE <222> (6) .. (6) <223> Xaa is absent, Asn or Gin <220>

<221> MISC FEATURE <222> (7) .. (7) <223> Xaa is Gin or Glu <220>

<221> MISC_FEATURE <222> (13) .. (13) <223> Xaa is His, Asp, Pro or Glu <220>

<221> MISC FEATURE <222> (19) .. (19) <223> Xaa is Tyr, Asp, Gin, His, Arg or Glu <220>

<221> MISC_FEATURE <222> (27) .. (27) <223> Xaa is Phe or His <220>

<221> MISC FEATURE <222> (28) .. (28) <223> Xaa is Phe or His <220>

<221> MISC_FEATURE <222> (29) .. (29) <223> Xaa is absent, Tyr, His, Thr, Gly or Asp <220>

<221> MISC_FEATURE <222> (30) .. (30) <223> Xaa is absent, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser or Glu <220>

<221> MISC_FEATURE <222> (31) .. (31) <223> Xaa is absent, Pro, His, Gly or Asp <220>

<221> MISCFEATURE <222> (32) .. (32) <223> Xaa is absent, Lys or Gin <220>

<221> MISC_FEATURE <222> (33) .. (33) <223> Xaa is absent or Thr <220>

<221> MISC_FEATURE <222> (34) .. (34) <223> Xaa is absent or Leu <220>

<221> MISC_FEATURE <222> (35) .. (35) <223> Xaa is absent or Glu <400> 2

Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Leu Cys Gly Ser Xaa Leu Vai Glu 15 10 15

Ala Leu Xaa Leu Vai Cys Gly Glu Arg Gly Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30

Xaa Xaa Xaa 35 <210> 3 <211> 21 <212> PRT <213> Artificial <220>

<223> Modified Insulin A chain <220>

<221> MISC_FEATURE <222> (8) .. (8) <223> Xaa is Thr or His <220>

<221> MISCFEATURE <222> (12) .. (12) <223> Xaa is Ser, Asp or Glu <220>

<221> MISC FEATURE <222> (13) .. (13) <223> Xaa is Leu, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser or Glu <220>

<221> MISC FEATURE <222> (14) .. (14) <223> Xaa is Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser or Glu <220>

<221> MISC FEATURE <222> (15) .. (15) <223> Xaa is Gin, Asp or Glu <220>

<221> MISC FEATURE <222> (18) .. (18) <223> Xaa is Asn, Lys or Gin <220>

<221> MISC FEATURE <222> (21) .. (21) <223> Xaa is Asn or Gin <400> 3

Gly lie Vai Glu Gin Cys Cys Xaa Ser lie Cys Xaa Xaa Xaa Xaa Leu 15 10 15

Glu Xaa Tyr Cys Xaa <210> 4 <211> 30 <212> PRT <213> Artificial <220>

<223> Modified Insulin B chain <220>

<221> MISCFEATURE <222> (1) .. (1) <223> Xaa is Phe or Glu <220>

<221> MISC_FEATURE <222> (3) .. (3) <223> Xaa is Asn or Gin <220>

<221> MISC FEATURE <222> (4) .. (4) <223> Xaa is Gin or Glu <220>

<221> MISC FEATURE <222> (10) .. (10) <223> Xaa is His, Asp, Pro or Glu <220>

<221> MISC_FEATURE <222> (16) .. (16) <223> Xaa is Tyr, Asp, Gin, His, Arg or Glu <220>

<221> MISC_FEATURE <222> (24) .. (24) <223> Xaa is Phe or His <220>

<221> MISC_FEATURE <222> (26) .. (26) <223> Xaa is absent, Tyr, His, Thr, Gly or Asp <220>

<221> MISC_FEATURE <222> (27) .. (27) <223> Xaa is absent, Thr, Asn, Asp, Gin, His, Lys, Gly, Arg, Pro, Ser or Glu <220>

<221> MISC_FEATURE <222> (28) .. (28) <223> Xaa is absent, Pro, His, Gly or Asp <220>

<221> MISCFEATURE <222> (29) .. (29) <223> Xaa is absent, Lys or Gln <220>

<221> MISC_FEATURE <222> (30) .. (30) <223> Xaa is absent or Thr <400> 4

Xaa Vai Xaa Xaa His Leu Cys Gly Ser Xaa Leu Vai Glu Ala Leu Xaa

10 15

Leu Vai Cys Gly Glu Arg Gly Xaa His Xaa Xaa Xaa Xaa Xaa

25 30

Claims (9)

1. Human insulin analog, characterized by the fact that the amino acid at position A12 is Glu, and / or the amino acid at position A13 is His, Asn or Glu, and / or the amino acid at position A14 is Glu, Asp or His, 5 and / or the amino acid at position A15 is Glu; and the amino acid at position B25 is His, and / or the amino acid at position B26 is Gly or Asp, and / or the amino acid at position B27 is Glu, Lys or Gly, and / or the amino acid at position B28 is Gly or Asp. 2. Human insulin analogue according to claim 1, 10 characterized by the fact that the amino acid at position A14 is Glu, Asp or His, the amino acid at position B25 is His, and that the insulin analogue optionally further comprises at least one additional mutation, wherein said additional mutation at the position of amino acid A8 is His and / or the position of amino acid A21 is Gly , and / or the position of the amino acid B10 is Glu 15 or Asp, and / or the position of the amino acid B16 is Glu or His, and / or the position of the amino acid in B27 is deleted, and / or the position of the amino acid in B28 is deleted, and / or the position of the amino acid in B29 is deleted, and / or the position of the amino acid in B30 is deleted. 3. Human insulin analogue according to claim 1 20 or 2, characterized by the fact that the additional mutations are A21 Gly, B16 Glu or His, desB27, desB28, desB29 and / or desB30. Human insulin analogue according to any one of claims 1 to 3, characterized by the fact that the amino acid at position A14 is Glu, Asp or His, and the amino acid at position B25 is His and the amino acid 25 in position B30 is deleted. 5. Human insulin analogue according to claim 4, characterized by the fact that A14 is Glu or Asp. 6. Human insulin analog, according to claim 1, characterized by the fact that it is selected from the group consisting of: Human insulin A8H, A14E, B1E, B16E, B25H, B27E, desB30; human insulin A8H, A14E, B1E, B25H, B27E, desB30; human insulin A8H, A14E, B1E, B25H, desB30; Petition 870180047259, of 06/04/2018, p. 17/22 human insulin A8H, A14E, B10E, B25H, desB30; human insulin A8H, A14E, B16E, B25H, desB30; human insulin A8H, A14E, B25H, B27E, desB30; human insulin A8H, A14E, B25H, desB30; 5 human insulin A8H, B10D, B25H; human insulin A12E, A14E, B25H, desB30; human insulin A13E, A14E, B25H, desB30; human insulin A13H, A14E, B25H, desB30; human insulin A13N, A14E, B25H, desB30; 10 human insulin A14D, B25H, desB30; human insulin A14E, B1E, B16E, B25H, B27E, desB30; human insulin A14E, B1E, B25H, B27E, desB30; human insulin A14E, B1E, B25H, desB30; human insulin A14E, B1E, B27E, desB30; 15 human insulin A14E, B10E, B25H, desB30; human insulin A14E, B16H, B25H, desB30; human insulin A14E, B25H; human insulin A14E, B25H, B26D, desB30; human insulin A14E, B25H, B26G, B27G, B28G, desB30; 20 human insulin A14E, B25H, desB27, desB28, desB29, desB30; human insulin A14E, B25H, desB27, desB30; human insulin A14E, B25H, B27E, desB30; human insulin A14E, B25H, B27R, desB30; human insulin A14E, B25H, B28D, desB30; 25 human insulin A14E, B25H, desB30; human insulin A14E, B27E, desB30; human insulin A14E, B28D, desB30; human insulin A14H, B10E, B25H, desB30; human insulin A14H, B16H, B25H, desB30; and 30 human insulin A14H, B25H, desB30. 7. Human insulin analogue according to any one of claims 1 to 6, characterized by the fact that it is for use as a Petition 870180047259, of 06/04/2018, p. 18/22 drug in the treatment or prevention of type 2 diabetes and type 1 diabetes. 8. Pharmaceutical composition, characterized in that it comprises a human insulin analog as defined in any one of claims 1 to 6, and a pharmaceutically acceptable carrier. Process for the preparation of a pharmaceutical composition, characterized in that it comprises mixing a human insulin analog, as defined in any one of claims 1 to 6, with pharmaceutically acceptable substances and / or excipients. Petition 870180047259, of 06/04/2018, p. 19/22 1/2 Proteolytic stability of insulin analogs in relation to chymotrypsin 120 100 Intact insulin (%) Time (min) at 37 ° C)